Dna molecules and methods

ABSTRACT

The present application discloses a DNA molecule comprising a modified Group II intron which does not express the intron-encoded reverse transcriptase but which contains a modified selectable marker gene in the reverse orientation, wherein the marker gene comprises a Group I intron in forward orientation of causing expression in a bacteria cell of the class Clostridia and wherein the DNA molecule comprises sequences that allow for the insertion of the RNA transcript of the Group II intron in the chromosome of a bacterial cell of the class Clostridia. A method of introducing a nucleic acid molecule into a site of a DNA molecule in a bacterial cell of the class Clostridia is also provided. The DNA molecule and the method are useful for making mutations  Clostridium  app.

The present invention relates to DNA molecules and methods using themolecules for introducing mutations into DNA in a Gram positivebacterial cell, particularly a cell of the class Clostridia.

The class Clostridia includes the orders Clostridiales, Halanaerobialesand Thermoanaerobacteriales. The order Clostridiales includes the familyClostridiaceae, which includes the genus Clostridium.

Clostridium is one of the largest bacterial genera. It is composed ofobligately anaerobic, Gram-positive, spore formers. Certain members maybe employed on an industrial scale for the production of chemical fuels,eg., Clostridium thermocellum and Clostridium acetobutylicum. Thislatter clostridial species, together with other benign representatives,additionally has demonstrable potential as a delivery vehicle fortherapeutic agents directed against cancer. However, the genus hasachieved greatest notoriety as a consequence of those members that causedisease in humans and domestic animals, eg, Clostridium difficile,Clostridium botulinum and Clostridium perfringens.

Despite the tremendous commercial and medical importance of the genus,progress either towards their effective exploitation, or on thedevelopment of rational approaches to counter the diseases they cause,has been severely hindered by the lack of a basic understanding of theorganisms' biology at the molecular level. This is largely a consequenceof an absence of effective genetic tools.

In recent years, the complete genome sequences of all of the majorspecies have been determined from at least one representative strain,including C. acetobutylicum, C. difficile, C. botulinum and C.perfringens. In other bacterial species such knowledge can act as aspringboard for more effective disease management or for the generationof strains with improved process properties. A pivotal tool in suchundertakings is the ability to rationally integrate DNA into the genome.Such technology may be employed: (i) to generate specific mutants as ameans of ascribing function to individual genes, and gene sets, as anessential first step towards understanding physiology and pathogenesis;(ii) to insertionally inactivate regulatory or structural genes as ameans of enhancing the production of desirable commercial commodities,and; (iii) to stably introduce genetic information encoding adventitiousfactors. However, there are currently no effective integration vectorsfor mutational studies in any Clostridium sp. and the ability toinsertionally inactivate genes in the genus remains woefully inadequate.

Previous attempts to make mutants in Clostridium sp. relied onhomologous recombination between an integration vector and the hostchromosome In C. perfringens strain 13, C. beijerinckii NCIMB 8052, C.acetobutylicum ATCC 824 and C. difficile CD37, replication-minusplasmids carrying regions of the host chromosome have been shown tointegrate into the genome via homologous recombination (Shimizu et al(1994) J. Bacteriol. 176: 1616-23; Wilkinson and Young (1994) Microbiol.140: 89-95; Green et al (1996) Microbiol. 142: 2079-2086; Liyanage et al(2001) Appl. Environ. Microbiol. 67: 2004-2010). In the case of C.beijerinckii and C. difficile, vectors were mobilized from E. colidonors. In C. perfringens and C. acetobutylicum, plasmids wereintroduced by transformation. Integrants arose in C. beijerinckii atfrequencies of 10⁻⁶ to 10⁻⁷ per recipient, which represented some twoorders of magnitude lower than the transfer frequency observed (10⁻⁴ to10⁻⁵) with replication proficient plasmids (Wilkinson and Young, 1994,supra). In the case of C. difficile, no indication of the frequenciesattained was reported (Liyanage et al, 2001, supra). In C.acetobutylicum, integrants arose at a frequency of 0.8 to 0.9 ‘colonies’per μg of DNA (Green et al, 1996, supra). In the above integrants,plasmid sequences at the target site were flanked by two directlyrepeated copies of the DNA segment directing integration. As aconsequence, they were segregationally unstable, e.g., losses per 30generations of between 1.8 to 3.0×10⁻³ for C. acetobutylicum (Green etal, 1996, supra) and between 0.37 to 1.3×10⁻³ for C. beijerinckii(Wilkinson and Young, 1994, supra).

It follows that integrants resulting from allelic exchange arepreferred. Accordingly, double crossover mutants were sought andobtained in C. perfringens (Awad et al (1995) Mol. Microbiol. 15:191-202; Bannam et al (1995) Mol. Microbiol. 16: 535-551. However,allelic exchange only proved possible through the inclusion of ratherlong (3.0 kb) regions of homology on either side of the antibioticresistance gene employed to inactivate the target gene. Furthermore,even with this provision, the isolation of mutants proved highlyvariable (i.e., plc mutants were only obtained in 2 of 10 independentexperiments), and many mutants can take up to 6 months to isolate, whileothers may never be isolated at all. Rare integration events could bedetected in C. perfringens as a consequence of the high frequency withwhich DNA can be transformed into this organism. Attempts to generatedouble crossover mutants in other clostridial species have beenunsuccessful.

To date the generation of mutants in a range of clostridial species,other than C. perfringens, using classical homologous recombination hasproven difficult. Thus, only five mutations have ever been made in C.acetobutylicum. Four (butK, CAC3075; pta, CAC1742; aad, CACP0162, and;solR, CACP061) were made by single cross-over integration of areplication deficient plasmids (Green et al., 1996, supra; Green andBennett (1996) Appl. Biochem. Biotechnol. 213, 57-58; Harris et al(2002) J. Bacteriol. 184, 3586-3597) while a fifth in spo0A (CAC2071)was isolated by a strategy which attempted, but did not succeed, in thegeneration of a mutant by reciprocal exchange using areplication-defective plasmid (Nair et al (1999) J. Bacteriol. 181,319-330). Similarly, the generation of only three directed mutants hasbeen reported in C. difficile. One mutant (gldA, CD0274) was generatedusing a replication-deficient plasmid (Liyanage et al, 2001, supra)although this event appeared to be lethal and mutant cells could not bepropagated. The other two genes inactivated (rgaR, CD3255 and rgbR,CD1089) arose following the introduction of a replication-defectiveplasmid carrying internal fragments of the two structural genes(O'Connor et al (2006) Mol. Microbiol. 61, 1335-1351). These latterplasmids were apparently introduced with “some difficulty” and whilstintegrants were isolated, no isolation frequencies were noted. Indeed,an assessment of the efficiencies of the mutagenesis procedurespreviously used in both organisms is difficult to make, as no indicationof the frequency with which mutants are generated is generallypresented. In the case of C. acetobutylicum it is acknowledged (Thomaset al, (2005) Metabolic engineering of soventogenic clostridia. In:Dürre, P. Handbook on Clostridia, CRC Press. pp 813-830) to be “lessthan one transformant per μg plasmid DNA”. Moreover, as the majority ofthese mutants are made by single cross-over insertion, they are unstabledue to plasmid excision. For example, Southern blotting of the C.difficile rgaR mutant revealed the presence of “looped out”,independently replicating plasmid in some cells in the population(O'Connor et al, 2006, supra).

Increasingly, technologies are being devised which capitalise on thesystems involving mobile genetic elements to bring about more effectivemodification of bacterial genomes. The Group II intron L1.LtrB ofLactococcus lactis is an element that mediates its own mobility throughthe action of an intron-encoded reverse transcriptase (LtrA) and theexcised lariat RNA. Furthermore, it may be re-targeted to virtually anydesired DNA sequence through modification of the intron RNA (Guo et at(2000) Science 289: 452-457; Mohr et at (2000) Genes Dev. 14: 559-573).Thus, by appropriately mutating individual bases in the 15 bp region ofthe intron involved in targeting, Karberg et al (Nature Biotech. (2001)19: 1162-1167) were able to direct the insertion of the element intodistinct, defined positions within several different E. coli genes atfrequencies of between 0.1 to 22%. Disruption of one of these genes,thyA, gives rise to clones that are naturally trimethoprim resistant.Thus, integrants could be selected for by culturing in the presence oftrimethoprim. Integrants in other genes were identified by screeningindividual colonies for the presence of the L1.LtrB intron. The plasmidused to disrupt the thyA gene in E. coli was also used to disrupt thethyA gene in S. flexneri and in S. typhimurium. Trimethoprim resistantcolonies were obtained at a frequency of 1% and 0.3% respectively.

The Group II intron L1.LtrB of Lactococcus lactis was used to generateknock-outs in the plc gene of C. perfringens (Chen et al (2005) ApplEnviron Microbiol. 71: 7542-7). A chloramphenicol resistant plasmidcontaining, inter alia, a modified L1.LtrB intron designed to target theplc gene was electroporated into C. perfringens. Transformants wereselected on chloramphenicol and were tested for the presence of theinsertion in the plc gene by PCR. Of 38 colonies tested, most werenegative for the insertion but two colonies contained both wild-type andintron-inserted plc gene. The latter colonies were deemed to have arisenfrom a single transformed bacterium, which gave rise to progeny in whichthe insertion occurred and progeny in which the insertion did not occur.Bacteria from these mixed colonies gave rise to pure clones, 10% ofwhich contained intron-inserted plc gene. Thus, insertion mutants wereidentified via two rounds of screening without the need for selectionfor growth on an antibiotic, other than selection on chloramphenicol fortransformation. In fact, the lack of any introduction of an antibioticresistance gene into the chromosome was identified as a particularadvantage of the method. In particular, the authors envisaged that themethod could be used to construct multiple gene disruptions in the samebacterial cell using the same shuttle plasmid carrying differentmodified L1.LtrB introns. The frequency of transfer to C. perfringens ishigh, some two orders of magnitude greater than other Clostridialspecies. Moreover, the gene knockout (in plc) gives rise to an easilydetected phenotype, which may be visualised readily on agar plates.

Yao et al (RNA (2006) 12: 1-11) used L1.LtrB to disrupt genes withoutselection in Staphylococcus aureus. A cadmium-induced promoter was usedto direct expression of the L1.LtrB intron in S. aureus; induction withcadmium was beneficial to obtaining insertion mutants in one gene. Whenmutants were made in another gene, all colonies tested positive forinsertion of the intron in the absence of cadmium.

Zhong et al (Nucleic Acids Res. (2003) 31: 1656-64) described a methodof positively selecting for re-targeting of the Group II introninvolving inserting into the Group II intron a“retrotransposition-activated selectable marker” or RAM consisting of atrimethoprim (Tp) resistance cassette containing the td intron of phageT4. The Tp resistance gene encodes a type II dihydrofolate reductase.The td intron is a Group I intron, i.e. a self-catalytic RNA-elementwhich, in its correct orientation, can splice itself from an RNAtranscript in which it is located. The orientation in which td isinserted into Tp^(R) is such that when the gene is transcribed, theelement is not spliced. Thus, the mRNA remains mutant, and the proteinrequired for Tp resistance is not produced. When the Group II element istranscribed into RNA, during re-targeting, the opposite strand of theRAM is now present in an RNA form. Under these circumstances the tdelement is orientated correctly, and is spliced. As a consequence, whenthe Group II element retargets to the chromosome, the Tp^(R) gene haslost its td insertion, and is now functional. As a consequence, cells inwhich successful re-targeting has taken place are Tp resistant. They maytherefore be directly selected. The method was used in Escherichia colicells.

Clostridial species are frequently resistant to trimethoprim, making theuse of a RAM based on a Tp resistance cassette unworkable. For instancein the study of Swenson et al (1980) Antimicrob. Agents Chemother. 18:13-19 the vast majority of the isolates tested were resistant.Resistance is also common in the non-pathogenic, industrially usefulstrains. Indeed, the intrinsic resistance of C. cellulolyticum forms thebasis of the conjugation method used for gene transfer experiments inJennert et al (2000) Microbiology. 146: 3071-80.

A kit for performing gene knockouts (principally in E. coli) based on aRAM consisting of a kanamycin resistance (Km^(r)) cassette is marketedas “TargeTron™ Gene Knockout System” by Sigma-Aldrich. Clostridium spp.are naturally resistant to kanamycin, so kanamycin resistance cannot beused as a selection marker in Clostridium.

The inability to make defined gene knock-outs in Clostridial genomes, byreciprocal marker exchange, is a major impediment to the commercialexploitation of members of the class Clostridia, and particularly thegenus Clostridium. It impinges on all areas. Thus, the application ofmetabolic engineering to generate industrial stains with improvedfermentation characteristics presently cannot be contemplated (eg C.acetobutylicum and the Acetone-Butanol fermentation process; strainscarrying chromosomally located therapeutic genes useful in cancertherapy cannot be generated (a prerequisite for clinical trials, eg C.sporogenes and Clostridial-Directed Enzyme Prodrug Therapy); andfundamental information on pathogenic mechanisms, an essential firststep in the formulation of effective countermeasures, is being severelyimpaired (eg C. difficile and hospital-acquired infections).

The listing or discussion of a prior-published document in thisspecification should not necessarily be taken as an acknowledgement thatthe document is part of the state of the art or is common generalknowledge.

The inventors have devised DNA molecules and methods which allow for theefficient insertion of DNA into the genome of Clostridium spp and otherbacteria of the class Clostridia, thereby allowing the targeted mutationof genes in the genome.

A first aspect of the invention provides a DNA molecule comprising:

-   -   a modified Group II intron which does not express the        intron-encoded reverse transcriptase but which contains a        modified selectable marker gene in the reverse orientation        relative to the modified Group II intron, wherein the selectable        marker gene comprises a region encoding a selectable marker and        a promoter operably linked to said region, which promoter is        capable of causing expression of the selectable marker encoded        by a single copy of the selectable marker gene in an amount        sufficient for the selectable marker to alter the phenotype of a        bacterial cell of the class Clostridia such that it can be        distinguished from the bacterial cell of the class Clostridia        lacking the selectable marker gene; and    -   a promoter for transcription of the modified Group II intron,        said promoter being operably linked to said modified Group II        intron; and

wherein the modified selectable marker gene contains a Group I intronpositioned in the forward orientation relative to the modified Group IIintron so as to disrupt expression of the selectable marker; and

wherein the DNA molecule allows for removal of the Group I intron fromthe RNA transcript of the modified Group II intron to leave a regionencoding the selectable marker and allows for the insertion of said RNAtranscript (or a DNA copy thereof) at a site in a DNA molecule in abacterial cell of the class Clostridia.

Group II introns are mobile genetic elements which are found ineubacteria and organelles. In nature, they use a mobility mechanismtermed retrohoming, which is mediated by a ribonucleoprotein (RNP)complex containing the intron-encoded reverse transcriptase (IERT) andthe excised intron lariat RNA. It is believed that the excised intronRNA inserts directly into one strand of a double-stranded DNA targetsite by a reverse splicing reaction, while the IERT alsosite-specifically cleaves the opposite strand and uses the 3′-end of thecleaved strand for target DNA-primed reverse transcription (TPRT) of theinserted intron RNA. As a result, the intron (and any nucleic acidcarried in a modified intron) are inserted into the target DNA. The TPRTsystem requires only the IERT and the excised intron RNA (see Saldanhaet al (1999) Biochemistry 38, 9069-9083). Details of Group II intronsare found in Karberg et al (2001) Nature Biotechnology 19, 1162-1167,incorporated herein by reference, and in references cited therein.

The IERT is also known in the art as the intron-encoded protein (IEP).The IEP (IERT) has reverse transcriptase activity as well asendonuclease and maturase activities which allow a copy of the intron tobe inserted into DNA.

The process of cleaving the DNA substrate and inserting nucleic acidmolecules involves base pairing of the Group II intron RNA of the RNPcomplex to a specific region of the DNA substrate. Additionalinteractions occur between the intron-encoded reverse transcriptase andregions in the DNA substrate flanking.the recognition site. Typically,the Group II intron RNA has two sequences, EBS1 and EBS2, that arecapable of hybridizing with two intron RNA-binding sequences, IBS1 andIBS2, on the top strand of the DNA substrate. Typically, the Group IIintron-encoded reverse transcriptase binds to a first sequence elementand to a second sequence element in the recognition site of thesubstrate. Typically, the Group II intron RNA is inserted into thecleavage site of the top strand of the DNA substrate. The first sequenceelement of the recognition site is upstream of the putative cleavagesite, the IBS1 sequence and the IBS2 sequence. The first sequenceelement comprises from about 10 to about 12 pairs of nucleotides. Thesecond sequence element of the recognition site is downstream of theputative cleavage site and comprises from about 10 to about 12nucleotides.

As denoted herein, nucleotides that are located upstream of the cleavagesite have a (−) position relative to the cleavage site, and nucleotidesthat are located downstream of the cleavage site have a (+) positionrelative to the cleavage site. Thus, the cleavage site is locatedbetween nucleotides −1 and +1 on the top strand of the double-strandedDNA substrate. The IBS1 sequence and the IBS2 sequence lie in a regionof the recognition site which extends from about position −1 to aboutposition −14 relative to the cleavage site.

Typically, EBS1 is located in domain I of the Group II intron RNA andcomprises from about 5 to 7 nucleotides that are capable of hybridizingto the nucleotides of the IBS1 sequence of the substrate.

Typically, EBS2 is located in domain I of the Group II intron RNAupstream of EBS1 and comprises from about 5 to 7 nucleotides that arecapable of hybridizing to the nucleotides of IBS2 sequence of thesubstrate.

In order to cleave the substrate efficiently, it is preferred that thenucleotide or sequence, which immediately precedes the first nucleotideof EBS1 of the Group II intron RNA, be complementary to the nucleotidesat +1 in the top strand of the substrate.

The modified Group II intron contained in the DNA molecule of theinvention does not express the IERT. Preferably, the Group II introndoes not contain a functional open reading frame for the IERT.Preferably, domain IV of the Group II intron, which typically containsthe IERT is partially deleted such that it does not contain the IERT.

Various Group II introns which may be useful in the practice of theinvention are known. These include bacterial introns such as theeubacterial introns reviewed in Dia and Zimmerly (2002) Nucleic AcidsRes. 30: 1091-1102, and also include mitochondrial and chloroplastintrons referred to in Zimmerly, Hausner and Wu (2001) Nucleic AcidsRes. 29: 1238-1250. It is preferred if the Group II intron is theLactococcus lactis L1.LtrB intron (Mohr et al (2000) supra). The IERT inthis Group II intron is the LtrA protein. The aI1 and aI2 nucleotideintegrases of Saccharomyces cerevisiae are also suitable.

Another alternative is the Group II intron from the clostridialconjugative transposon Tn5397 (Roberts et al (2001) J. Bacteriol. 183:1296-1299).

The LtrA RNP complex comprises an excised, wild-type or modified excisedGroup L1.LtrB Group II intron RNA of the Lactococcus lactis LtrB gene,hereinafter referred to as the “L1.LtrB intron” RNA, and a wild-type ormodified L1.LtrB intron-encoded reverse transcriptase, referred to asthe LtrA protein. The EBS1 of the L1.LtrB intron RNA comprises 7nucleotides and is located at positions 457 to 463. The EBS1 sequence ofthe wildtype L1.LtrB intron RNA has the sequence 5′-GUUGUGG (SEQ ID No.1). The EBS2 of the L1.LtrB intron RNA comprises 6 nucleotides and islocated at positions 401 to and including 406, The EBS2 sequence of thewild-type L1.LtrB intron RNA has the sequence 5′AUGUGU (SEQ ID No. 2).

The Group II intron in the DNA molecule of the invention has beenmodified to include a modified selectable marker gene. A selectablemarker gene is any gene which confers an altered phenotype in abacterial cell in which it is expressed, compared to the bacterial cellin which it is not expressed. The modified selectable marker gene ismodified (compared to the unmodified selectable marker gene) bycontaining a Group I intron which disrupts the expression of theselectable marker. The term “unmodified selectable marker gene” includesa gene comprising a promoter and a coding region of a gene, where thepromoter is not the promoter of the naturally occurring gene.“Unmodified selectable marker” also includes where the promoter is thepromoter of the naturally occurring gene. Further details of themodification of the selectable marker gene are described below but, inessence, the presence of the Group I intron prevents the expression ofthe selectable marker but, upon excision of the Group I intron, theresulting nucleic acid (ie unmodified selectable marker gene) is able toexpress the selectable marker, Preferably, the selectable marker gene islocated in domain IV of the Group II intron.

It will be appreciated that the Group I intron may be positioned at anylocation within the selectable marker gene as long as expression of theselectable marker is prevented by the presence of the Group I intron. Itwill be appreciated that the Group I intron may be positioned, forexample, within the promoter, such as between the −10 and −35 elementsof the promoter, between the promoter and the coding region or in thecoding region.

The selectable marker gene containing the Group I intron (ie themodified selectable marker gene) may be considered to be aretrotransposition activated marker (RAM).

Group I introns are self-splicing introns which may or may not requireauxiliary factors such as proteins in order to be excised. Various GroupI introns which may be useful in the practice of the invention are knownincluding bacteriophage introns (Sandegran and Sjöberg (2004) J. Biol.Chem. 279: 22218-22227), and Tetrahymena Group I intron (Roman (1998)Biochem. 95: 2134-2139). It is preferred that the Group I introns do notrequire auxiliary factors in order to be excised. It is preferred if theGroup I intron is the td Group I intron from Phage T4 (EhrenMan et al(1986) Proc. Natl. Acad. Sci. USA 83: 5875-5879).

It will be appreciated that the orientation of the various componentswithin the DNA molecule is very important. Thus, from FIG. 2 it will beseen that the modified selectable marker gene is present within theGroup II intron in the reverse orientation to the Group II intron. Also,the Group I intron which is present within the modified selectablemarker gene in a reverse orientation to the selectable marker gene butin the same forward orientation as the Group II intron. If the Group Iintron were in the same orientation as the selectable marker gene, theintron would be able to excise from the mRNA transcript of theselectable marker gene and the phenotype conferred by the selectablemarker would be present irrespective of whether the Group II introncontaining the selectable marker had retargeted to the chromosome.Therefore, the Group I intron and the selectable marker gene must be inopposite orientations.

If the selectable marker gene were in the same orientation as the GroupII intron, following the above logic, the Group I intron would have tobe in the opposite orientation to the Group II intron. However, in thisorientation, it would not excise from the mRNA traneript and so, even ifthe Group II intron did retarget to the chromosome, there would be noselectable phenotype.

Only when the various components are orientated as shown in FIG. 2 willretargeting of the Group II intron to the chromosome be necessary andsufficient for expression of the selectable marker phenotype.

When the DNA molecule of the invention is used to introduce a nucleicacid molecule into a site of a DNA molecule in a bacterial cell of theclass Clostridia (as is described in more detail below), the Group Iintron is removed from the RNA transcript produced from the modifiedGroup II intron to leave a region encoding the selectable marker, andthe RNA transcript (or a DNA copy thereof) is introduced into a site ina DNA molecule in a bacterial cell of the class Clostridia. In this way,the nucleic acid introduced into a DNA molecule in a bacterial cell ofthe class Clostridia has a selectable marker gene which is able toexpress the selectable marker in the bacterial cell.

In a preferred embodiment, the modified Group II intron is flanked byexons, which exons allow splicing of an RNA transcript of the Group IIintron.

The promoter of the selectable marker gene is capable of causingexpression of the selectable marker when it is encoded by a single copyof the selectable marker gene in an amount sufficient for the selectablemarkers to alter the phenotype of a bacterial cell of the classClostridia such that it can be distinguished from the bacterial cell ofthe class Clostridia lacking the selectable marker gene. For example,the promoter may be one which, when present in a single copy in thebacterial chromosome, and when in operable linkage with the codingregion of the selectable marker, expresses the selectable marker in adetectable amount. The promoter of the selectable marker gene is onewhich is functional in a bacterial cell of the class Clostridia andcauses adequate expression when present in a single copy as describedabove. It is preferred that the promoter is functional in a Clostridiumsp. Suitable promoters include the fdx gene promoter of C. perfringens(Takamizawa et al (2004) Protein Expression Purification 36: 70-75); theptb, thl and the adc promoters of C. acetobutylicum (Tummala et al(1999) App. Environ. Microbiol. 65: 3793-3799) and the cpe promoter ofC. perfringens (Melville, Labbe and Sonenshein (1994) Infection andImmunity 62: 5550-5558) and the thiolase promoter from C. acetobutylicum(Winzer et al (2000) J. Mol. Microbiol. Biotechnol. 2: 531-541).Preferably, the promoter of the selectable marker gene is the promoterof the thl gene of C. acetobutylicum.

To test whether a promoter is likely to be effective as a promoter of aselectable marker of the invention, a spliced variant of the RAM (ieencoding the selectable marker since the Group I intron has beenremoved) may be placed under its transcriptional control and introducedinto the Clostridia to be targeted at a low copy number, preferablyequivalent to the copy number of the chromosome. This can be achieved byusing a low copy number plasmid, such as the low copy number derivativesof plasmid pAMB1 described in Swinfield et al (1990) Gene. 87:79-90 ormore ideally using a conjugative transposon and the method described inMullany et al (Plasmid (1994) 31: 320-323) and Roberts et al (JMicrobiol Methods (2003) 55: 617-624). To achieve the latter, thespliced RAM together with the promoter under evaluation may be clonedinto a vector that is unable to replicate in a Gram-positive bacteriumbut which carries an antibiotic resistance gene (eg catP) and a segmentof DNA derived from a conjugative transposon, such as Tn916. The plasmidis then transformed into a Bacillus subtilis cell that carries theappropriate conjugative transposon in its genome (Tn916), andtransformants selected on plates containing chloramphenicol. As theplasmid cannot replicate, the only way that chloramphenicol resistantcolonies can arise is if the plasmid integrates into the genome as aconsequence of homologous recombination between Tn916 and the region ofhomology carried by the plasmid. This results in a transposon::plasmidcointegrate carrying the spliced RAM and promoter under test that islocated in a single copy in the genome. The Bacillus subtilistransconjugant obtained may now be used as a donor in a conjugation withthe Clostridia to be targeted. In these matings, transfer of thetransposon::plasmid cointegrate into the Clostridia recipient can beselected on the basis of acquisition of resistance to thiamphenicol.Once obtained, transconjugants may be tested for the resistance encodedby the RAM, eg., erythromycin.

The promoter for regulating the transcription of the modified Group IIintron may be any suitable promoter which is functional in a bacterialcell of the class clostridia. The promoter may be a constitutivepromoter or an inducible promoter. An inducible promoter may bederepressed such that it drives expression in a constitutive fashion. Inparticular experiments described in the Examples, the inventors foundthat regulated expression of the modified Group II intron confers noadvantage in allowing for a high intron insertion frequency compared toconstitutive expression. However, in other situations, it may be usefulto be able to regulate expression of the modified Group II intron. Aperson of ordinary skill can perform experiments to determine whether aparticular promoter is suitable to allow for a satisfactory introninsertion rate.

Girbal et al (2003) Appl. Environ. Microbial. 69: 4985-4988 describe apreferred xylose-inducible promoter in C. acetobutylicum, which is basedon the Staphylococcus xylosus xylose operon promoter-repressorregulatory system. Suitable inducible promoters are IPTG orxylose-inducible. Conveniently, for example when the DNA molecule is foruse in Clostridial cells, the promoter is the promoter region of the C.pasteurianum ferredoxin gene under the control of the lac operatorregion of the E. coli lac operon. Conveniently, the DNA molecule furthercomprises the lacI gene of E. coli.

A promoter for regulating the transcription of the modified Group IIintron may be a constitutive promoter. The skilled person willappreciate that in general all promoters are regulated under onecondition or another, even if such conditions are not known. Therefore,we intend “constitutive promoter” to be interpreted broadly to encompassa promoter that is active in the Clostridial cells under the normalculture conditions employed in the retargeting protocol, without theneed for addition of an agent to activate expression driven by thepromoter. Promoters of genes that are essential to primary metabolismmay be suitable “constitutive promoters”. For example, the thiolasepromoter, thl, described in the Examples may be a suitable promoter.Other suitable promoters are the C. acetobutylicum promoters hbd, crt,etfA, etfB amd bcd (Alsaker and Papoutsakis (2005) J Bacteriol187:7103-7118). Promoters suggested as being suitable for drivingexpression of the modified selectable marker in the RAM may also besuitable.

The use of an inducible promoter allows transcription of the Group IIintron containing the selectable marker gene interrupted by the Group Iintron (which may be termed a RAM) to be switched off followingretargeting of the RAM to the bacterial chromosome. When the RAM istranscribed from the inducible promoter, expression of the selectablemarker is ineffective. This may be because of duplex formation betweenthe transcripts of the coding strand transcribed from the chromosome andthe non-coding strand transcribed from the DNA molecules.

The DNA molecule of the invention preferably is capable of replicationin a bacterial cell of the class Clostridia. More preferably, it iscapable of conditional replication. Conveniently, the DNA moleculecontains a suitable origin of replication and any necessary replicationgenes to allow for replication in the Gram-positive bacterial cell (iesuitable rep genes). Preferably, the DNA is a plasmid. Alternatively,the DNA may be linear or it may be filamentous phage like M13.Conveniently, the DNA molecule is a shuttle vector which allows forreplication and propagation in a Gram-negative bacterial cell such asEscherichia coli and for replication in a Gram-positive cell,particularly a cell of the class Clostridia and more particularly of thegenus Clostridium. Additionally or alternatively, the DNA molecule ofthe invention contains a region which permits conjugative transfer fromone bacterial cell to a bacterial cell of the class Clostridia. It isparticularly preferred if the DNA molecule contains a region whichpermits conjugative transfer between E. coli and a bacterium of theclass Clostridia, and more particularly of the genus Clostridium. Forexample, the DNA molecule may contain the oriT (origin of transfer)region, including the traJ gene.

Methods of transformation and conjugation in Clostridia are provided inDavis, I, Carter, G, Young, M and Minton, NP (2005) “Gene Cloning inClostridia”, In: Handbook on Clostridia (Durre P, ed) pages 37-52, CRCPress, Boca Raton, USA.

The selectable marker may be any suitable selectable marker which can beexpressed in and used to select a cell of the class Clostridiacontaining the selectable marker. Suitable selectable markers includeenzymes that detoxify a toxin, such as prodrug-converting enzymes.Selectable markers also include a prototrophic gene (for use in acorresponding auxotrophic mutant). Preferably, the selectable marker isone which gives a growth advantage to the bacterial cell of the classClostridia in which it is expressed. Thus, typically, under a givengrowth condition the bacterial cell which expresses the selectablemarker is able to grow (or grow more quicldy) compared to an equivalentcell that does not express the selectable marker.

Convenient selectable markers include antibiotic resistance factors.Thus, suitably, the selectable marker gene is a gene which confersantibiotic resistance on a bacterial cell of the class Clostridia.

Not all antibiotic resistance genes can be used in all cells of theclass Clostridia. For example, Clostridium sp. are naturally resistantto kanamycin, and are frequently resistant to trimethoprim. Thus, it ispreferred that the selectable marker gene is not a kanamycin resistancegene or a trimethoprim resistance gene particularly when the bacterialcell is of the genus Clostridium. Suitable antibiotic resistance genesfor use in Clostridial cells, such as Clostridium sp., includeerythromycin resistance genes (such as Erm) and chloramphenicolresistance genes (such as catP). Another suitable antibiotic resistancegene is tetM, for example tetM from the Enterococcus faecalis Tn916conjugative transposon (Roberts et al (2001) Microbiol. 147: 1243-1251).Another suitable antibiotic resistance gene, widely used in bacteria ofthe class Clostridia, is spectinomycin adenyltransferase, aad(Charpentier et al (2004) Appl. Environ. Microbiol. 70, 6076-6085).

The methods and DNA molecules of the invention may also be used toinvestigate genes the function of which is not known. For example, theDNA molecule of the invention may be adapted to contain a uniqueoligonucleotide sequence referred to as a tag which will be introducedinto the DNA in the cell of the class Clostridia. Conveniently, aplurality of DNA molecules of the invention are produced, eachcontaining a different tag sequence. When the DNA inserts into thebacterial chromosome, the tag is present in the genomic DNA and may bedetected for example by amplification by hybridising to a labelledoligonucleotide probe, a portion of which has a sequence complementaryto a portion of the tag. Suitable tags, probes and methods of amplifyingand hybridising are described in Hensel et al (1995) Science 269:400-403. A plurality of mutants may be generated by the method of theinvention in which each has the DNA inserted into a different gene, andeach may be identified by its unique tag. Typically, each differentretargeting nucleic acid contains targeting portions which direct it toa different gene in the DNA of the cell of the class Clostridia. Theplurality of mutants may be introduced into an environment for a periodof time. Mutants may then be recovered from the environment. The abilityof individual tags to be detected in the recovered pool of mutants givesan indication of whether a particular mutant has been able to grow orsurvive as well as other mutants. In this way, genes that are requiredfor growth or survival in the environment may be identified. Hensel etal (1995; supra) used a similar approach to identify virulence genes inSalmonella.

In a modification of the above method, DNA molecules of the inventionhaving the same tag but different randomised Group II intron targetingportions and corresponding exon sequences may be generated, pooled andused to make bacterial mutants. Group II introns with randomisedtargeting portions are described in WO 01/29059. Many of the DNAmolecules may be unable to insert anywhere in the bacterial genome.However, some may be able to insert at an unknown location in thebacterial genome governed by the sequence of the targeting portions. Asufficiently large pool of DNA molecules of the invention may be used inthe method such that one or more colonies are obtained in which the DNAhas inserted into the chromosome. A single clone may be selected. Theprocess may be repeated for a pool of DNA molecules of the inventionhaving a different unique tag, to obtain another single mutant bacterialclone with a unique tag. In this way, a plurality of bacterial mutants,each with a unique tag are generated. The plurality of mutants may beexposed to an environment as described above, to identify particularmutants that are compromised for growth or survival in that environment.A mutant identified from such a screen may then be characterised todetermine in which gene the DNA has inserted.

Further details of genes encoding modified selectable markers whichcontain a Group I intron which disrupts the expression of the selectablemarker are given below.

The selectable marker gene or its coding region may be associated withregions of DNA for example flanked by regions of DNA that allow for theexcision of the selectable marker gene or its coding region followingits incorporation into the chromosome. Thus, a clone of a mutantClostridial cell expressing the selectable marker is selected andmanipulated to allow for removal of the selectable marker gene.Recombinases may be used to excise the region of DNA. Typically,recombinases recognise particular DNA sequences flanking the region thatis excised. Cre recombinase or FLP recombinase are preferredrecombinases. Alternatively, an extremely rare-cutting restrictionenzyme could be used, to cut the DNA molecule at restriction sitesintroduced flanking the selectable marker gene or its coding region. Apreferred restriction enzyme is I-SceI.

A mutant bacterial cell from which the selectable marker gene has beenexcised retains the Group II intron insertion. Accordingly, it has thesame phenotype due to the insertion with or without the selectablemarker gene. Such a mutant bacterial cell can be subjected to a furthermutation by the method of the invention, as it lacks the selectablemarker gene present in the RAM.

Although the modified Group II intron in the DNA molecule of theinvention does not express the IERT, conveniently the DNA moleculecontains in another location a gene which is able to express the IERT.

Where the Clostridial cell into which the Group II intron is to beinserted uses a different genetic code from the Group II intron and itsassociated Group II intron-encoded reverse transcriptase, it ispreferred that the sequence of the Group II intron-encoded reversetranscriptase is modified to comprises codons that correspond to thegenetic code of the host cell.

A particularly desirable embodiment of the invention is wherein themodified Group II intron comprises targeting portions. Typically, thetargeting portions allow for the insertion of the RNA transcript of themodified Group II intron into a site within a DNA molecule in theClostridial cell. Typically, the site is a selected site, and thetargeting portions of the modified Group II intron are chosen to targetthe selected site. In a preferred embodiment, the selected site is inthe chromosomal DNA of the Clostridial cell. Typically, the selectedsite is within a particular gene, or within a portion of DNA whichaffects the expression of a particular gene. Insertion of the modifiedGroup II intron at such a site typically disrupts the expression of thegene and leads to a change in phenotype.

Genes may be selected for mutation for the purposes of metabolicengineering. For example, in organisms such as Thermoanaerobacteriumsaccharolyticum, or other members of the class Clostridia which have asimilar metabolism, deletion of lactate dehyrogenase andphosphotransacetylase to prevent formation of lactate and acetate,respectively, could be used to elevate levels of ethanol (Desai et al,(2004) Appl Microbiol Biotechnol. 65: 600-5). In solventogenicclostridia, such as Clostridium acetobutylicum and Clostridiumbeijerinckii, specific deletions may be made to the genes encoding theenzymes responsible for solvent and acid production as a means ofmaximising acetone and butanol (see Jones and Woods (1986) MicrobialRev. 50: 484-524). Thus, strains could be generated that produce onlyacetone or butanol, by elimination of enzymes responsible for productionof acetate (phosphotransacetylase and or acetate kinase), butyrate(phosphotransbutyrylase and or butyrate kinase), butanol (butanoldehydrogenase A and/or butanol dehydrogenase B) and/or acetone(acetyoacetate decarboxylase and/or acetoacetyl-CoA transferase).Moreover, the fermentative ability of such strains could be extended bygene addition into the chromosome, such that new substrates could bedegraded (sugars, lignocellulose, hemicellulose, etc.) and/or new endproducts made (isopropanol, 1,3-propanediol, etc.).

Genes may be selected for mutation in order to determine the role oftheir encoded products in virulence, a prerequisite to the developmentof vaccines and other countermeasures. In C. difficile, for example, therelative roles of toxin A and toxin B (CdtA and CdtB) remain to beestablished (Bongaerts and Lyerly (1994) Microbial Pathogenesis 17:1-12) due to a previous inability to generate isogenic mutants. Certainstrains (Perelle et al (1994) Infect Immun. 65: 1402-1407) also producean actin-specific ADP-ribosyltransferase CDT (CdtA and CdtB). Otherfactors undoubtedly contribute to virulence, particularly the initialcolonisation process. The participation of a number of gene products hasbeen proposed (Tasteyre et al (2001) Infect Immun 69: 7937-7940; Calabiet al (2002) Infect Immun 70: 5770-5778; Waligora et al (2001) InfectImmun 69: 2144-2153), including those involved in adhesion, the S-layerproteins (Sp1A) and motility (FliC and FliD). Definitive proof of theinvolvement of these factors in disease through the generation ofmutants has until now not been possible.

The DNA sequences of the genomes of many bacteria of the classClostridia are known. For example, the DNA sequences of the genomes ofC. acetobutylicum (ATCC 824 (GenBank Accession No AE001437), C.difficile (GenBank Accession No AM180355), C. tetani E88 (GenBankAccession No AE015927) and C. perfringens strain 13 (GenBank AccessionNo BA000016) and C. botulinum are known. The sequence of a C. sporogenesgenome is partially known and is very similar to the sequence of the C.botulinum genome. From this information, sites for insertion are readilyidentified, for example within open reading frames. It is preferred ifthe DNA molecule of the invention contains a modified Group II intronwhich contains targeting portions which targets the RNA transcript ofthe modified Group II intron (or a DNA copy thereof) into a gene in thegenome of one of these bacterial species.

As described above, Group II introns naturally contain regions whichtarget the intron to a specified sequence in target DNA. Because therecognition site of the DNA substrate is recognized, in part, throughbase pairing with the excised Group II intron RNA of the RNP complex, itis possible to control the site of nucleic acid insertion within the DNAsubstrate. This may be done by modifying the EBS 1 sequence, the EBS2sequence or the δ sequence, or combinations thereof. Such modified GroupII introns produce RNP complexes that can cleave DNA substrates andinsert nucleic acid molecules at new recognition sites in the genome.For example, by reference to the L1.LtrB Group II intron of Lactococcuslactis illustrated in FIGS. 1A and 1B the EBS1, EBS2 and δ are modifiedto permit base pairing of the RNA transcript of the modified Group IIintron with a target site. Rules for DNA target-site recognition byL1.LtrB Group II intron which enable retargeting of the intron tospecific DNA sequences are described in Mohr et al (2000) Genes &Development 14, 559-573, incorporated herein by reference.Computer-aided design of targeting portions are also described inPerutka et al (2004) J. Mol. Biol. 336, 421-429, incorporated herein byreference.

WO 01/29059 to the Ohio State University Research Foundation,incorporated herein by reference, describes a selection-based approachin which the desired DNA target site is cloned into a recipient vectorupstream of a promoterless tet^(R) gene. Introns that insert into thatsite are selected from a combinatorial donor library having randomizedtargeting portions (EBS and δ) and IBS exon sequences. The modifiedL1.LtrB intron contains a heterologous promoter, such that when itinserts into the target site in the recipient vector, the tet^(R) geneis transcribed and the bacterial cell containing the vectors may beselected for. The sequence of the modified intron may be determined byPCR. Thus, a modified Group II intron DNA may be isolated that allowsfor insertion into the target DNA site within a Clostridial cell.

In the case of the L1.LtrB Group II intron, it is thought that theinteraction of the δ region with a δ′ region of the target DNA is notcritical to efficient retrohoming of the Group II intron. However, theinteractions between EBS2 and EBS1 in the intron RNA and IBS2 and IBS1in the target DNA are more important.

When the Group II intron excises from the RNA transcript, it is believedthat it transiently base-pairs with portions of the flanking exon RNA.In particular, the EBS2 and EBS1 regions base-pair with the IBS2 andIBS1 regions of the 5′ exon respectively. Therefore, it is preferredthat the IBS2 and IBS1 region of the 5′ exon is modified so as topromote base-pairing with the modified EBS2 and EBS1 regions of theintron RNA. This facilitates efficient excision of the Group II intronfrom its RNA trancript.

Modification of the EBS2 and EBS1δ sites and the IBS2 IBS1 site mayconveniently be performed using any suitable site directed mutagenesismethods known in the art, for example oligonucleotide-directedmutagenesis or PCR-based methods.

Typically, the DNA molecule of the invention is able to express anantibiotic resistance marker which is different to the selectablemarker. For example, if the selectable marker gene is a first antibioticresistance gene the DNA includes a second antibiotic resistance gene. Itis particularly preferred if both antibiotic resistance genes are oneswhich give rise to antibiotic resistance in Clostridial cells. Forexample, the selectable marker gene in the DNA molecule may be anerythromycin resistance gene and the DNA molecule may further contain achloramphenicol resistance gene (or vice versa). When the DNA moleculeis for use in a Clostridium sp. it is particularly preferred that anyantibiotic resistance genes are selected from erythromycin resistancegenes (eg ermB) or chloramphenicol resistance genes (eg catP).

It will be appreciated that although it is convenient for the DNAmolecule of the invention to itself contain a gene which is able toexpress the IERT, this may be provided on a separate DNA molecule. Thus,a further aspect of the invention provides a kit of parts comprising aDNA molecule of the first aspect of the invention and a separate DNAmolecule which is able to express the IERT. Typically, the DNA moleculesare plasmids, preferably compatible plasmids. It will be appreciatedthat the kit may further contain a DNA molecule (typically a plasmid)which is able to express the lac repressor protein. This is useful inthe situation where the DNA molecule of the invention comprises anIPTG-inducible promoter which is operatively linked to the Group IIintron, but when the DNA molecule of the invention does not include thelacI gene.

A third aspect of the invention provides a method of introducing anucleic acid molecule into a site of a DNA molecule in a bacterial cellof the class Clostridia, the method comprising the steps of:

-   -   (i) providing a bacterial cell of the class Clostridia with the        DNA molecule of the invention and a DNA molecule capable of        expressing a Group II intron-encoded reverse transcriptase; and    -   (ii) culturing the bacterial cell under conditions which allow        for removal of the Group I intron from the RNA transcript of the        modified Group II intron and the insertion of said RNA        transcript containing the selectable marker gene (or a DNA copy        thereof) into said site.

Preferably, the bacterial cell of the class Clostridia is cultured underconditions which allow for expression of the selectable marker.Typically, the bacterial cell of the order Clostridia into which nucleicacid has been introduced at a site of a

DNA molecule within the cell (ie mutated cell) is selected based on analtered phenotype conferred by the selectable marker.

Conveniently, the selectable marker is an antibiotic resistance markerand the mutated Clostridial cell is selected on the basis of its abilityto grow in the presence of the relevant antibiotic.

Conveniently, the selected cell is cloned and a single clone of cells isobtained.

A further aspect of the invention provides a method of targeting anucleic acid molecule to a selected site of a DNA molecule in abacterial cell of the class Clostridia, the method comprising providinga bacterial cell of the class Clostridia with a DNA molecule of theinvention in which the modified Group II intron comprises targetingportions and a DNA molecule capable of expressing a Group IIintron-encoded reverse transcriptase; and culturing the bacterial cellunder conditions which allow removal of the Group I intron from the RNAtranscript of the modified Group II intron and the insertion of said RNAtranscript (or DNA copy thereof) containing the selectable marker geneinto said selected site.

It will be appreciated that in this way it is possible to make sitedirected mutations in DNA (such as the genome) of a bacterial cell ofthe class Clostridia, such as a Clostridium spp.

Mutant bacterial cells of the class Clostridia obtained by the methodsof the invention are also part of the invention.

It will be appreciated that with respect to all aspects of the inventionit is preferred that the bacterial cell of the class Clostridia is aClostridium spp. It is particularly preferred if the Clostridial cell isC. thermocellum or C. acetobutylicum or C. difficile or C. botulinum orC. perfringens or C. sporogenes or C. beijerinckii or C. tetani or C.cellulyticum or C. septicum. The Clostridial cell may alternatively byThermoanaerobacteria saccharolyticum, an important species forindustrial ethanol production. By the term “Clostridia”, we also includeRoseburia, such as Roseburia intestinalis, which is a probioticbacterium. Thus, preferably, the selectable marker gene in the DNAmolecule of the invention is a gene which can be used for selection inthese species (eg an erythromycin resistance gene or a chloramphenicolresistance gene or a tetracycline resistance gene or a spectinomycinresistance gene). Also preferably, the DNA molecules of the inventioncontain origins of replication and any necessary replication genes whichallow for replication in these bacterial species.

A particular feature of the invention is that the modified selectablemarker gene is one which contains a Group I intron which disruptsexpression of the selectable marker. The selectable marker is one whichmay be expressed in and used for selection in a bacterial cell of theclass Clostridia, particularly a Clostridium cell.

It is particularly preferred that the selectable marker is an antibioticresistance io gene which can be used for selection in a Clostridium spp.

A further aspect of the invention provides a DNA molecule comprising amodified erythromycin-resistance gene which contains a Group I intron.

A further aspect of the invention provides a DNA molecule comprising amodified chloramphenicol-resistance gene which contains a Group Iintron.

A further aspect of the invention provides a DNA molecule comprising amodified tetracycline-resistance gene which contains a Group I intron.

A further aspect of the invention provides a DNA molecule comprising amodified spectinomycin resistance gene which contains a Group I intron.

The invention also includes these DNA molecules present in a host cell,for example an E. coli cell or a cell of the class Clostridia.

Preferably the Group I intron is present in the opposite orientation tothe antibiotic resistance gene.

The Group I intron may be present anywhere within the antibioticresistance gene, for example within the coding region thereby disruptingtranslation, or upstream of the coding region thereby disruptingtranscription or translation.

The Group I intron is present within the antibiotic resistance gene in aform whereby when the intron is transcribed it is able to excise(splice) itself from the RNA transcript.

Any autocatalytic RNA which can self-splice out of a larger RNA in anorientation-dependent manner could substitute for a Group I intron inthe present invention. Suitably, an “IStron” may be used, which isbelieved to be a fusion of a Group I intron and an IS element(Haselmayer et al (2004) Anaerobe 10: 85-92; Braun et al (2000) Mol.Microbiol. 36: 1447-1459).

For the avoidance of doubt, for the purposes of all aspects of theinvention any autocatalytic RNA which can self-splice out of a largerRNA in an orientation-dependent manner is considered to be a Group Iintron, whether or not it requires auxiliary factors. Preferably theGroup I intron does not require auxiliary factors.

It is preferred that the Group I intron does not encode anintron-encoded protein such as an intron-encoded reverse transcriptase.This feature prevents the excised Group I intron RNA from re-insertingat another site within the bacterial genome.

It is noted that, typically, the splicing of Group I introns (such asthe td intron of Phage T4) is reliant on exon sequences flanking thepoint of insertion. Thus, the modified selectable marker genes of theinvention (and in particular the modified antibiotic resistance geneswhich encode erythromycin resistance and chloramphenicol resistance andtetracycline resistance and spectinomycin resistance of this aspect ofthe invention) contain the Group I intron inserted in a position wherebyit is flanked by suitable exon sequences that allow the Group I intronto splice out of the RNA transcript and wherein the resulting splicedtranscript (or DNA copy thereof) encodes a functional selectable marker(such as functional erythromycin resistance or functionalchloramphenicol resistance). Suitable flanking sequences are known forGroup I introns. For example, for the Phage T4 td Group I intron, theintron is typically preceded by a G residue (ie present 5′ of theintron) and the intron is typically followed by the sequence5′-ACCCAAGAGA-3′ (SEQ ID No. 3)(ie present 3′ of the intron).Alternatively, the intron may be followed by the sequence5′-ACCCAAGAA-3′ (SEQ ID No. 4).

In a preferred embodiment of the invention, the coding region of theselectable marker (such as the erythromycin or chloramphenicol ortetracycline or spectinomycin resistance genes) contains suitablesequences which flank the intron. In relation to the td intron, and thecombined 5′ and 3′ flanking sequence 5′-GACCCAAGAGA-3′ (SEQ ID No. 5)this is able to code for several amino acid sequences depending on thereading frame (as explained in more detail in the examples).

In Frame 1, it encodes the amino acid sequence DPRD/E (SEQ ID No. 6); inFrame 2 it encodes the amino acid sequence R/GPKR (SEQ ID No. 7) and inFrame 3 it encodes the amino acid sequence “X″TQE″Z” (SEQ ID No. 8)where X can be any of G, E, A, V, L, S, W, P, Q, R, M, T or K and “Z”can be any of K, S, R, I, M, T or N.

Thus, in a preferred embodiment, the coding region of the selectablemarker gene encodes a portion of peptide with the above amino acidsequence.

In a further preferred embodiment, the exon sequence 3′ of the intron ispresent in an appropriate reading frame at the 5′ end of the codingsequence of the selectable marker so that, in the absence of the intron,the coding sequence encodes a functional selectable marker whichcontains a linker peptide at the N-terminus of the selectable markerpolypeptide.

The linker peptide is typically a peptide of 4 to 20, preferably 4 to15, typically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acidresidues, a portion of which are encodable by the exon coding sequencesflanking the intron. The presence of the linker peptide does notinterfere substantially with antibiotic resistance activity. In otherwords, the polypeptide produced from expression of the nucleic acidmolecule produced when the Group I intron has been excised hasantibiotic resistance activity.

Alternatively, the Group I intron flanking sequence may be disposed sothat the insertion of the Group I intron disrupts transcription of theselectable marker gene. For example, it may be located between the −35and −10 elements of the promoter.

In a further alternative, the Group I intron flanking sequence may bedisposed so that the insertion of the Group I intron disruptstranslation of the selectable marker gene. For example, it may belocated between the ribosome binding site and the start codon.

It will be appreciated that the DNA molecules of the invention may bemade using standard molecular biological techniques as described inSambrook et al, “Molecular cloning: A laboratory manual”, 2001, 3^(rd)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The invention will now be described with reference to the followingnon-limiting Examples and Figures.

FIG. 1. A: Secondary structure model of L1.LtrB group II intron. Thepredicted secondary structure consists of six domains (I-VI). TheEBS2/IBS2, EBS1/IBS1 and δ-δ′ interactions between the intron andflanking exons in unspliced precursor RNA are indicated by broken lines.In the un-modified L1.LtrB intron, the open reading frame encoding theLtrA protein is present in the non-structural loop indicated as domainIV. B: Mechanism of DNA target site recognition by L1.LtrB group IIintron. The LtrA protein binds to the L1.LtrB group II intron RNAforming a ribonucleoprotein complex. The intron splices out of thepre-mRNA, liberating the ribonucleoprotein as a particle. Theribonucleoprotein particle locates target DNA sequences within the cell.The target DNA sequence of the unmodified ribonucleoprotein is anintronless copy of the ltrB gene, the sequence of which is depicted (SEQID No. 9). The intron RNA is inserted into the insertion site within thetop strand (IS). The bottom strand is then cleaved at the cleavage site(CS) and the LtrA primes from the cut DNA and reverse-transcribes theintron RNA. Host repair activities complete the integration process.Recognition of the target is mediated by a combination of interactionsbetween LtrA and nucleotides in the target sequence, and between EBS2and EBS1 in the intron RNA and complementary sequences IBS2 and IBS 1 inthe target sequence. The most important of the nucleotides recognised bythe LtrA protein are indicated by grey shading.

FIG. 2. Positive Selection of retargeting nucleic acid-derived mutants.A. Transcription of the selectable marker gene from the plasmid-locatedretargeting nucleic acid does not result in resistance, because the mRNAproduced retains the td group I intron insertion and the expression ofthe selectable marker gene is therefore disrupted. The td element cannotsplice out of the mRNA because it has been transcribed in the wrongorientation. B. L1.LtrA group II intron RNA production is induced byaddition of IPTG, causing transcription from Clostridial promoter fac.The td group I intron within the selectable marker gene is transcribedin the correct orientation and the td RNA splices out of the RNAproduced. C. The L1.LtrA RNA and the selectable marker gene are insertedinto the target site in the chromosome. The selectable marker gene doesnot contain the td group I intron and therefore the expression of theselectable marker gene is not disrupted. The cells therefore exhibit thephenotype associated with expression of the selectable marker, and maybe selected accordingly.

FIG. 3. Inducible expression from pMTL5401Fcat in C. sporogenes and C.acetobutylicum.

(a) The E. coli/Clostridium shuttle plasmid pMTL5401Fcat. (b) A clone ofC. sporogenes or (c) C. acetobutylicum containing pMTL5401Fcat was grownto early exponential growth phase and the CAT activity in cell lysatesmonitored after induction with 1 mM IPTG (▪) or without induction (▴).

FIG. 4. Sequences suitable for a selectable marker gene for successfulsplicing of the td group I intron. The required amino acid sequences(SEQ ID Nos. 6-8, in any of the three translation reading frames, areshown above the nucleotide sequences (SEQ ID Nos. 132-134). Amino acidsat position ‘X’ could be either G, E, A, V, L, S, W, P, Q, R, M, T or K.At position ‘Z’ they could be K, S, R, I, M, T or N.

FIG. 5. RAM functionality added to the ermB gene using a linker.

(a) A linker containing the td intron and its exons was inserted betweenthe ermB ORF and its promoter (SEQ ID No. 10), preventing expression oferythromycin-resistance. Splicing of the td intron out of the reversestrand yields a modified ermB gene (SEQ ID No. 11) that encodes afunctional protein with 12 additional amino acids at its N-terminus (SEQID No. 12). The ermB promoter of ErmBtdRAM1 is replaced by the thlpromoter in ErmBtdRAM2.

(b) PCR using various templates and primers ErmB-Pro-F3 and ErmB-R1,which flank the td intron in ErmBtdRAM1. Lane 1: ErmBtdRAM1 DNA; Lane 2:ErmBtdRAM1 SE DNA; Lane 3: cDNA synthesised from RNA isolated from cellscontaining pMTL20lacZTTErmBtdRAM1 after IPTG induction; Lane 4: the sameRNA preparation before cDNA synthesis.

(c) PCR using various templates and primers Thio-F1 and ErmB-R1, whichflank the td intron in ErmBtdRAM2. Lane 1: C. sporogenes spo0A mutantgenomic DNA; Lane 2: pMTL007::Csp-spo0A-249s plasmid DNA; Lane 3: C.sporogenes wild-type genomic DNA; Lane 4: water.

FIG. 6. Features and sequence of ErmBtdRAM1

ErmBtdRAM1 sequence (SEQ ID No. 13)

FIG. 7. Direct evidence that the ErmBtd RAM1 is spliced in E. coli.

To test that the td group I intron has been spliced from ErmBtdRAM1following induction of the group II intron RNA expression, RNA wasprepared from cells expressing pMTL20lacZTTErmBtdRAM1. RT-PCR wasperformed using primers that flank the td site of insertion. In controlreactions, the same primers were used to amplify ErmBtdRAM1 and SplicedEquivalent SE DNA by PCR. Lane 1: DNA markers; lane 2, PCR of ErmBtdRAM1; lane 3, PCR of ErmBtd RAM1 SE; lane 4, RT-PCR on total RNA fromcells containing pMTL20lacZTTErmBtdRAM1, and; lane 5 RT-PCR negativecontrol.

FIG. 8. Construction of a multicloning site in pBRR3

Sequences of the cloning sites of pBRR3-LtrB (SEQ ID No. 14) andpCR2.1-TOPO plasmids (SEQ ID No. 15). The multicloning site fragmentdepicted (SEQ ID No. 16) was inserted into a cleaved pBRR3-LtrB to makepBRR3-MCS1 depicted (SEQ ID No. 17), containing restriction sites foundin the pCR2.1-TOPO plasmid.

FIG. 9. Sequences of the thl and thl2 promoters

Sequences of thl (SEQ ID No. 18) and thl2 promoters (SEQ ID No. 19) areshown in comparison to a consensus promoter (SEQ ID No. 20). “x”indicates a nucleotide substitution compared to the consensus sequence.The spacing between the −10 and the −35 elements is indicated for eachsequence.

FIG. 10. Features and sequence of ErmBtdRAM2

ErmBtdRAM2 sequence (SEQ ID No. 21)

FIG. 11. Features and sequence of pMTL007

Plasmid map of the final clostridial retargeting system (the illustratedexample is a derivative modified to re-target lacZ) and sequence (SEQ IDNo. 22)

FIG. 12. Construction of pMTL5401F

Restriction sites used at each step are indicated. DNA end-blunting wasperformed using T4 DNA polymerase.

FIG. 13. Construction of pMTL5402F and pMTL5402F-lacZTTErmBtdRAM1

Restriction sites used at each step are indicated. DNA end-blunting wasperformed using T4 DNA polymerase.

FIG. 14. Construction of pMTL007

Restriction sites used at each step are indicated.

FIG. 15. Examples of mutant screening and characterisation.

(a) Plasmid pMTL007. (b, c) PCR was used to initially screen for thepresence of the intron insertion in the C. difficile spo0A gene usingthe intron-specific primer EBS Universal and gene-specific primerCd-spo0A-R2 (small arrows). Lane 1: water; Lane 2: C. difficile parentalstrain genomic DNA; Lane 3: pMTL007::Cdi-spo0A-178a plasmid DNA; Lanes4-6: DNA from three randomly-selected Em^(R) C. difficile clonesgenerated using pMTL007::Cdi-spo0A-178a. (d) Southern blots of the spo0Aand pyrF mutants of C. difficile using a probe to erinB. Hybridisationof this probe to the pre-existing (non-functional) chromosomal ermB ORFcauses a second band, also visible in the parental lanes. In the EcoRVdigest of the spo0A mutant, both bands are a similar size. (e)Equivalent Southern blot for C. acetobutylicum and (f) C. sporogenes.

FIG. 16. The spo0A mutants do not form spores.

Phase-contrast micrographs of the spo0A mutants and parental strains ofC. difficile, C. acetobutylicum and C. sporogenes grown on solid mediafor 14 days, 4 days or 3 days respectively. Mean sporulation frequenciesof three separate experiments are shown as percentages.

EXAMPLE 1 Development of an IPTG-Inducible ‘fac’ Promoter

The use of a E. coli/Clostridium shuttle vector (pMTL540F) carrying theartificial promoter ‘fac’ has previously been described. It was derivedby inserting the operator of the E. coli lacZ operon immediatelydownstream of the promoter of the C. pastueurianum ferredoxin gene (Foxet al (1996) Gene Ther. 3: 173-178). Although this promoter element wasused to direct the high level expression of heterologous genes inclostridia, regulated transcription has not been demonstrated. A new E.coli/Clostridium shuttle vector pMTL5401F was, therefore, constructedfeaturing the fac promoter, a lacI repressor gene under thetranscriptional control of the promoter of the C. acetobutylicumphosphotransbutyrylase (ptb) gene and the oriT region of plasmid RK2 tofacilitate conjugative transfer to C. sporogenes, C. botulinum and C.difficile. To test pMTL5401F, we inserted a promoterless copy of thepC194 cat gene, such that its transcription was under the control of thefac promoter in the resultant plasmid, pMTL5401Fcat (FIG. 3). We thenassayed for the enzyme activity of the cat gene product in the lysatesof C. sporogenes or C. acetobutylicum cells carrying pMTL5401Fcat, grownin the presence or absence of exogenous IPTG. Induction was observed inboth organisms, but while strong repression of transcription was evidentin C. sporogenes in the absence of IPTG (FIG. 3), a significant basallevel of expression was observed in C. acetobutylicum (FIG. 3). AlthoughpMTL5401F could be introduced into C. difficile, the pCB102 repliconfunctions relatively ineffectively in this clostridial host (Purdy et al(2002) Mol. Microbiol. 46: 439-452) and cannot support the growth of itstransconjugants in antibiotic-supplemented liquid culture. Therefore, anequivalent induction experiment could not be performed.

EXAMPLE 2 Development of ErmBtd as a Selectable Marker for Clostridia

Splicing of the td group I intron is reliant on exon sequences flankingthe point of insertion. The target site recognised by the Phage T4 tdgroup I intron is 5′-GACCCAAGAA-3′ (SEQ ID No. 23) and the introninserts after the intial ‘G’. However the td group I intron will alsoinsert at the site 5′-GACCCAAGAGA-3′ (SEQ ID No. 5) (Sigma AldrichTargeTron™ Gene Knockout System). Sequences of antibiotic genescurrently in use in Clostridia were evaluated for the presence of thesesequences but no genes incorporating either of these sequences wereidentified. If the splice site 5′-GACCCAAGAGA-3′ (SEQ ID No. 5) werepresent in a protein coding region, the amino acid sequence it wouldencode would depend on its reading frame. Amino acid sequences(corresponding to the three possible frames) that may be encoded by thesplice site are shown in FIG. 4. Screening of the protein sequences ofall proteins known to confer resistance on clostridia failed to identifya candidate protein containing any of the desired amino acid sequences.

Accordingly, a gene encoding a selectable marker was engineered suchthat it contained an insertion site for the td group I intron. This wasto form the basis of a Clostridial RAM. The native ermB gene of theEnterococcus faecalis plasmid pAMβ1, which confers resistance toerythromycin, was chosen as the selectable marker gene because this genehas been widely used in the construction of E. coli/Clostridium shuttlevectors (Dürre, P. Handbook on Clostridia. 2005. Taylor and Francis, CRCPress.)

A linker sequence was designed which contained the required splice site,and fused to the 5′ end of the coding region of the ermB gene, in effectextending the N-terminus of the protein by 12 amino acids (FIG. 5). Inthe design of this sequence, the chosen reading frame was that whichencoded amino acids that were as inert as possible, and soluble, tominimise risk of adversely affecting ErmB protein function. Frame 1(DPRD; SEQ ID No. 6) was the best option as it includes three chargedresidues (Asp −ve, Arg +ve) which should favour solubility. It was hopedthat the mixture of charges might help prevent a strong interaction withthe rest of the protein. The rest of the linker was composed of thesmall, inert residues Gly and Ala, with a single Ser to avoid a longstretch of hydrophobic residues which might reduce the protein'ssolubility. In addition, the nucleotide sequence chosen incorporatesclostridial codon usage, to minimise any potential expression problems.

Two constructs were assembled using SOEing PCR as described below, usingoligonucleotide primers indicated in Table 1 below. The ErmBtd RAM1 (themodified ermB gene containing the td intron inserted at the indicatedsite in FIG. 6 (SEQ ID No. 13), and the spliced equivalent (SE), inwhich the td intron is absent. ErmBtdRAM1 and ErmBtdRAM1 SE were eachcloned into the high copy plasmid pMTL5402F in the opposite orientationto the fac promoter (so that any resistance conferred is due to the RAMor SE's own promoter).

TABLE 1 Oligonucleotide primers SEQ Primer Sequence (5′-3′) ID NOErmB-Pro-F3 CTACGCGTGGAAATAAGACTTAGAAGCAA 24 ACTTAAGAGTGTG ErmB-Pro-RACAGAAGCACCAGCATCTCTTGGGTCCATGT 25 AATCACTCCTTCTTAATTACAAATTTTTAG CATClinker1- ACCCAAGAGATGCTGGTGCTTCTGGTGCTG 26 ErmB-F1GTATGAACAAAAATATAAAATATTCTCAA AACTTTTTAACGAGTG ErmB-R1GAACGCGTGCGACTCATAGAATTATTTCCT 27 CCCG ErmB-Pro-GGGGTAAGATTAACGACCTTATCTGAACAT 28 RB AATGCCATGTAATCACTCCTTCTTAATTACAAATTTTTAGCATC tdGpI-F1 GCATTATGTTCAGATAAGGTCGTTAATCTT 29 ACCCC tdGpI-R1CCAGAAGCACCAGCATCTCTTGGGTTAATT 30 GAGGCCTGAGTATAAG Thio-F1CTACTAGTACGCGTTATATTGATAAAAATA 31 ATAATAGTGGG Thio-R-RAMCCTTATCTGAACATAATGCCATATGAATCC 32 CTCCTAATTTATACGTTTTCTC

The ErmBtdRAM1 SE was made as follows. The ermB promoter wasPCR-amplified from pMTL5402F using primers ErmB-Pro-F3 and ErmB-Pro-RA.The ermB ORF was PCR-amplified from pMTL5402F using primerslinker1-ErmB-F1 and ErmB-R1. The PCR products were gel-purified and usedas templates in a SOEing PCR using the outer primers ErmB-Pro-F3 andErmB-R1. The PCR product encoding ErmBtdRAM1 SE was cloned intopCR2.1-TOPO. ErmBtdRAM1 SE was excised from pCR2.1::ErmBtdRAM1SE as aHindIII/XhoI fragment and ligated into pMTL5402F linearised with thesame enzymes. This placed ErmBtdRAM1 SE in the opposite orientation tothe fac promoter on the resulting plasmid pMTL5402F::ErmBtdRAM1SE.

The ErmBtdRAM1 construct was made as follows. The ermB promoter wasPCR-amplified from pMTL5402F using primers ErmB-Pro-F3 and ErmB-Pro-RB.The ermB ORF was PCR-amplified from pMTL5402F using primerslinker1-ErmB-F1 and ErmB-R1. The attenuated td group I intron and itsexons were PCR-amplified from pACD4K-C using primers tdGpI-F1 andtdGpI-R1. The PCR products were gel-purified and used as templates in a3-way SOEing PCR using the outer primers ErmB-Pro-F3 and ErmB-R1. ThePCR product encoding ErmBtdRAM1 was cloned into pCR2.1-TOPO. ErmBtdRAM1was excised from pCR2.1::ErmBtdRAM1 as a HindIII/XhoI fragment andligated into pMTL5402F linearised with the same enzymes.

E. coli carrying pMTL5402F::ErmBtdRAM1 was sensitive to erythromycin at500 and 125 μg/ml (no growth overnight at 37° C.). E. coli carryingpMTL5402F::ErmBtdRAM1SE was resistant to erythromycin at 500 and 125μg/ml (grew overnight at 37° C.). These experiments demonstrated thatthe modified ermB gene conferred resistance to erythromycin in E. coli,and equally important, that the insertion of td inactivates the gene.

EXAMPLE 3 Validation of the ErmBtd Selectable Marker in E. coil

The retargeting nucleic acid component of pACD4K-C was sub-cloned as aNaeI (blunt) fragment into pMTL20 (Chambers et al (1988) Gene 68:139-149) between HindIII and SmaI sites and the lacZ re-targeting regionagain shown to be able to knock-out the lacZ gene in the E. coli hostHMS174(DE3). Next the KanRAM in pMTL20lacZTT was replaced with ErmBtdRAM1 as MluI fragment. To test that the td group I intron was beingspliced from ErmBtd RAM1 following induction of group II intron RNAexpression, E. coli cells carrying pMTL20lacZTTErmBtdRAM1 were harvestedand RNA prepared. RT-PCR reactions were then undertaken using primersthat flank the td site of insertion. As a control, standard PCR wasperformed on ErmBtd RAM1 and ErmBtd RAM1 SE (the spliced equivalent ofErmBtd RAM1). As can be seen in FIG. 7, the predominant product obtainedfrom the IPTG induced RNA samples was of the smaller size correspondingto the SE gene. This clearly demonstrates that td is being spliced fromthe RNA of the modified ermB gene in ErmBtd RAM1.

Despite the fact that demonstrable splicing of ErmBtd RAM1 had beenshown to occur, no erythromycin resistant colonies were obtainedfollowing plating of the IPTG induced cells on agar media supplementedwith 500, 250 or 125 μg/ml erythromycin. It was not possible to reducethe concentration of antibiotic any further, as E. coli is naturallyresistant to lower levels of the antibiotic.

Failure to obtain erythromycin resistant colonies may have been due to acopy number effect. Thus, a single copy inserted in the genome may havebeen insufficient to raise resistance to the antibiotic above the usuallow level of resistance inherent to wild type E. coli. To test thispossibility, a DNA fragment fragment carrying ErmBtdRAM1 SE was ligatedto cleaved pACYC184, and the ligation mixture transformed into E. coliand plated on 2YT containing either tetracycline or erythromycin atthree different concentrations, 500, 250 and 125 μg/ml. Similar numbersof colonies grew on Erm125 and Tet, but several-fold less grew onErm250, and only a few grew on Erm500. This control experiment set thepractical limit for the screening of the inheritance of ErmBtdRAM1 SEwhen present on pACYC184 as being 125 μg/ml.

Having established the level of erythromycin needed to screen forErmBtdRAM1 SE in E. coli, a region of lacZ encompassing the targetingregion was PCR amplified with primers lacZ target-F(ACGAATTCCGGATAATGCGAACAGC-GCACGG; SEQ ID No. 33) and lacZ target-R(TGCGATCGCACCGCCGA-CGGCACGCTGATTG; SEQ ID No. 34), cloned intopCR2.1TOPO, and then subcloned into pACYC184, which is present atseveral copies in the E. coli cell. The re-targeting experiment was thenrepeated by introducing pMTL20lacZTTErmBtdRAM1 into E. coli cellscarrying pACYC184::lacZ. Following induction with IPTG, the cells wereplated onto media containing erythromycin. In contrast to the previousexperiment, appreciable numbers of resistant colonies were obtained. Theuse of appropriate primers in a diagnostic PCR confirmed thatre-targeting of the group II intron to the lacZ gene on pACYC184 hadtaken place. Therefore, when ErmBtdRAM1 SE is present as a single copy,expression of ErmB is insufficient to confer resistance to erythromycin,but when present in multiple copies, ErmB is expressed at a sufficientamount to confer the resistant phenotype.

EXAMPLE 4 Construction of a Clostridial Retargeting System Using theErmBtd Selectable Marker

Having established that ErmBtdRAM1 could substitute for the KanRAM inthe Sigma-Aldrich group II intron, the entire element, together with there-targeting region for lacZ, was subcloned from pMTL20lacZTTErmBtdRAM1(as HindIII/SacI and SacI/NheI fragments) into the clostridialexpression vector pMTL5402F (cleaved with HindIII-NheI) to givepMTL5402FlacZTTErmBtdRAM1. As a consequence, expression of the group IIintron was under the control of the fac promoter. Expression of thegroup II intron will be regulated by IPTG.

The ability of this vector to re-target the lacZ gene on pACYC184::lacZin E. coli was tested. Following IPTG induction and plating onerythromycin, successful re-targeting was demonstrated.

EXAMPLE 5 Determination of the Efficiency of ErmBtdRAM1 in Group IIIntron Retargeting

To assess whether ErmBtdRAM1 affects the frequency with which the groupII intron can retarget, compared to KanRAM, we undertook some mobilityassays using a two-plasmid system developed Karberg et al (2001, sup-a).Retargeting of the group II intron from pACD2, following IPTG-induction,to pBRR3-LtrB (which carries its natural target, LtrB) results inactivation of the Tet gene on the latter plasmid. Thus, individualretargeting events can be detected on the basis of acquisition ofresistance to Tetracycline.

Plasmid pACD2 was therefore modified by the insertion of either theErmBtdRAM1 or the KanRAM, into the vector's unique MluI site. These twoplasmids were then transformed into HMS174(DE3) cells containingpBRR3-LtrB—i.e. the recipient plasmid with the wild type targetsequence. After selection for the donor plasmid, cells were induced with500 μM IPTG for 1 hr, re-suspended in LB, allowed to recover for 1 hr,and then various dilutions were plated onto various selective plates.For those constructs containing a RAM, Tet^(R) colonies were firstre-streaked onto Tet plates and then again onto plates containing theappropriate antibiotic to test RAM splicing. Results are shown in Table2.

This experiment demonstrated that the KanRAM and ErmBtdRAM1 have asimilar effect on intron efficiency—presumably mainly due to theincreased size of the intron. Importantly, the data indicate that bothRAMs splice at similar efficiencies.

TABLE 2 Results of mobility assays Results Intron RAM Donor plasmidmobility efficiency* splicing efficiency† pACD2 (none ~10⁰    n/apACD2::KanRAM) ~10⁻³ 18/20 pACD2::ErmBtdRAM ~10⁻³ 18/20 *Intron mobilityefficiency = Tet^(R) colonies/Amp^(R) Cm^(R) colonies †RAM splicingefficiency = Kan^(R) or Erm^(R) re-streaked Tet^(R) colonies/allre-streaked Tet^(R) colonies

Splicing of neither RAM could be detected by antibiotic resistanceinitially, but only when re-streaked from Tet^(R) colonies.

EXAMPLE 6 Identification of effective Clostridial Re-Targeting Sequences

To evaluate retargeting of the ErmBtdRAM1, eight different test geneswere chosen from 3 different clostridial species. These were:Clostridium sporogenes pyrF, spo0A, codY, and SONO, Clostridiumdifficile pyrF (Genome Annotation No. CD3592) and spo0A (GenomeAnnotation No. CD1214), Clostridium aceotbutylicum pyrF (GenomeAnnotation No. CAC2652) and spo0A.

Each gene was analysed at http://www.sigma-genosys.com/targetron/, andsuitable changes to allow for re-targeting identified. Using appropriateprimers, the generation of appropriately modified Group II introns waseffected by performing a PCR as directed in the Sigma-Aldrich TargeTron™Gene Knockout System User Guide. Each PCR required unique IBS, EBS2 andEBS1d primers designed to modify the targeting portions of the Group IIintron or its 5′ exon, and the EBS Universal primer. The sequences ofthe target insertion sites for each gene and primers are given in Tables3 and 4 below.

TABLE 3 Predicted target insertion sites for retargeting nucleic acidsTarget insertion SEQ Target^(a) site sequence 5′-3′ ID No C. sporogenesGCTAGATTTGATAAAGAATTTAC 35 codY 417s TGATGAA-intron-GATTTAGT GTTAGCAC. difficile CAACGTATTGCTCTAGCCCTACC 36 pyrF 97a TTAAATA-intron-TGTCTACACTATCTT C. difficile ATCCATCTAGATGTGGCATTATT 37 spo0A 178aACATCTA-intron-GTATTAAT AAGTCCG C. sporogenes AATAGTATAGATATTACTCCTAT 38spo0A 249s GCCAAGG-intron-GTAATTGT TTTGTCT C. sporogenesGTAATTGTGGATATAGCTCTATA 39 pyrF 595s GGAGCAG-intron-TAGTTGGA TGTACAGC. acetobutylicum GAAATGTATGCTAAAGCTCACTT 40 pyrF 345sTGAAGGT-intron-GATTTTGA AGCGGAT C. acetobutylicumCCAACAGCGGATAAAACTATTAT 41 spoOA 242a TCTTGGA-intron-AGGTTTTC TGCATCTC. sporogenes ATCAAAGTAGATGAAATAGAAAG 42 SONO 492sAAAAGAT-intron-GATTTTTT AAAACTT ^(a)Target indicated as organism, ORFand insertion point. Target insertion sites were selected such thatintrons would be inserted after the indicated number of bases from thestart of the ORF, in either the sense (s) or antisense (a) orientation.

TABLE 4 Oligonucleotide primers used to generatePCR products for retargeting SEQ Primer Primer sequence 5′-3′ ID No.EBS Universal CGAAATTAGAAACTTGCGTTCAGTA 43 AAC Csp-codY-AAAAAAGCTTATAATTATCCTTATT 44 417s-IBS TACCGATGAAGTGCGCCCAGATAGG GTGCsp-codY- CAGATTGTACAAATGTGGTGATAAC 45 417s-EBS1dAGATAAGTCGATGAAGATAACTTAC CTTTCTTTGT Csp-codY-TGAACGCAAGTTTCTAATTTCGGTTG 46 417s-EBS2 TAAATCGATAGAGGAAAGTGTCTCdi-pyrF-97a- AAAAAAGCTTATAATTATCCTTACTA 47 IBSCCCTAAATAGTGCGCCCAGATAGGGT G Cdi-pyrF-97a- CAGATTGTACAAATGTGGTGATAACA 48EBS1d GATAAGTCTAAATATGTAACTTACCT TTCTTTGT Cdi-pyrF-97a-TGAACGCAAGTTTCTAATTTCGGTTG 49 EBS2 GTAGTCGATAGAGGAAAGTGTCT Cdi-spo0A-AAAAAAGCTTATAATTATCCTTATTA 50 178a-IBS TTCCATCTAGTGCGCCCAGATAGGGT GCdi-spo0A- CAGATTGTACAAATGTGGTGATAACA 51 178a-EBS1dGATAAGTCCATCTAGTTAACTTACCT TTCTTTGT Cdi-spo0A-TGAACGCAAGTTTCTAATTTCGGTTA 52 178a-EBS2 ATAATCGATAGAGGAAAGTGTCTCsp-spo0A- AAAAAAGCTTATAATTATCCTTACCT 53 249s-IBSATCCCAAGGGTGCGCCCAGATAGGGT G Csp-spo0A- CAGATTGTACAAATGTGGTGATAACA 54249s-EBS1d GATAAGTCCCAAGGGTTAACTTACCT TTCTTTGT Csp-spo0A-TGAACGCAAGTTTCTAATTTCGGTTA 55 249s-EBS2 TAGGTCGATAGAGGAAAGTGTCTCsp-pyrF-595s- AAAAAAGCTTATAATTATCCTTACTA 56 IBSTACGAGCAGGTGCGCCCAGATAGGGT G Csp-pyrF-595s- CAGATTGTACAAATGTGGTGATAACA57 EBS1d GATAAGTCGAGCAGTATAACTTACCT TTCTTTGT Csp-pyrF-595s-TGAACGCAAGTTTCTAATTTCGGTTT 58 EBS2 ATAGTCGATAGAGGAAAGTGTCTCac-pyrF-345s- AAAAAAGCTTATAATTATCCTTACAC 59 IBSTTCGAAGGTGTGCGCCCAGATAGGGT G Cac-pyrF-345s- CAGATTGTACAAATGTGGTGATAACA60 EBS1d GATAAGTCGAAGGTGATAACTTACCT TTCTTTG Cac-pyrF-345s-TGAACGCAAGTTTCTAATTTCGGTTA 61 EBS2 AGTGTCGATAGAGGAAAGTGTCT Cac-spo0A-AAAAAAGCTTATAATTATCCTTAATT 62 242a-IBS ATCCTTGGAGTGCGCCCAGATAGGGT GCac-spo0A- CAGATTGTACAAATGTGGTGATAACA 63 242a-EBS1dGATAAGTCCTTGGAAGTAACTTACCT TTCTTTGT Cac-spo0A-TGAACGCAAGTTTCTAATTTCGGTTA 64 242a-EBS2 TAATCCGATAGAGGAAAGTGTCTCsp-SONO- AAAAAAGCTTATAATTATCCTTAGAA 65 492s-IBSAGCAAAGATGTGCGCCCAGATAGGGT G Csp-SONO- CAGATTGTACAAATGTGGTGATAACA 66492s-EBS1d GATAAGTCAAAGATGATAACTTACCT TTCTTTGT Csp-SONO-TGAACGCAAGTTTCTAATTTCGATTC 67 492s-EBS2 TTTCTCGATAGAGGAAAGTGTCT

To ensure that the modified group II introns were capable of retargetingto the selected clostridial genes, experiments were first undertaken inE. coli using plasmid systems that were known to function effectively.The system utilised is a two-plasmid system developed by Karberg et al(2001) as described in Example 5. Using this system, the engineeredgroup II intron is placed on one plasmid (pACD2) and its target (in thiscase the cloned clostridial gene) is placed on a second plasmid (pBRR3).Retargeting of the group II intron from pACD2 to pBRR3 results inactivation of the Tet gene on the latter plasmid. Thus, individualretargeting events can be detected on the basis of acquisition ofresistance to Tetracycline. A portion of the bacteria are plated onnon-selective agar plates to give an indication of total viable bacteriaand a portion are plated on tetracycline-containing agar plates (Tetplates). The efficiency of retargeting is estimated based on theproportion of total viable bacteria that are resistant to tetracycline.

To facilitate subcloning of the target genes from pCR2.1/pCRII TOPOplasmids into pBRR3, a multiple cloning site was introduced intopBRR3-LtrB to make pBRR3-MCS1. This was done by insertion of amulticloning site fragment between the AatII and EcoRI sites ofpBRR3-LtrB, containing restriction sites found in the pCR2.1-TOPOplasmid. Sequences of the cloning sites are given in FIG. 8. Themulticloning site fragment depicted in FIG. 8 was made from MCS1aoligonucleotide CTCGAGGTACCATGCATAGGCCTGAGCTCA-CTAGTGCGGCCGCG (SEQ IDNo. 68) and MCS1b oligonucleotideAATTC-GCGGCCGCACTAGTGAGCTCAGGCCTATGCATGGTACCTCGAGACGT (SEQ ID No. 69).

Four retargeting nucleic acids (each intended for insertion in one of C.sporogenes genes pyrF, spo0A, codY, and SONO) were evaluated using thetwo-plasmid intron mobility assay. All four permitted far more efficientretargeting than anticipated. Consequently the dilutions chosen forplating on Tet plates were not ideal and were only just in range forcolony counts and therefore the efficiencies given may be less accuratethan if fewer bacteria had been plated. The next four retargetingnucleic acids (each intended for insertion in one of C. difficile pyrFand spo0A genes or C. aceotbutylicum pyrF and spo0A genes) wereevaluated for retargeting. Retargeting events were estimated by platingbacteria on Tet plates. In this initial experiment, SONO gave no Em^(R)colonies. Results are shown in Table 5.

TABLE 5 Results of intron mobility assay Intron mobility Donor plasmidRecipient plasmid efficiency* pACD2::Cs-spo0A-249s TR pBRR3::Cs-spo0AAS2 frag   ~15% pACD2::Cs-codY-417s TR pBRR3::Cs-codY AS2 frag   ~20%pACD2::Cd-pyrF-97a TR pBRR3::Cd-pyrF-97 target  ~100%pACD2::Cd-spo0A-178a TR pBRR3::Cd-spo0A-178 target   ~20%pACD2::Cs-pyrF-595s TR pBRR3::Cs-pyrF   ~2% pACD2::Ca-pyrF-345s TRpBRR3::Ca-pyrF  ~0.2% pACD2::Ca-spo0A-242a TR pBRR3::Cs-spo0A   ~20%pACD2::Cs-SONO-492s TR pBRR3::Cs-SONO″ ND *Intron mobility efficiency =Tet^(R) colonies/Amp^(R) Cm^(R) colonies, Cs—C. sporogenes, Ca—C.acetobutylicum, Cd—C. difficile. Numbers refer to the site of introninsertion relative to the start of the gene, in either the sense (s) orantisense (a) orientation. TR = retargeting nucleic acid

EXAMPLE 7 Evaluation of Retargeting Nucleic Acids in Clostridia

The first four new retargeting nucleic acids (each intended forinsertion in one of C. sporogenes genes pyrF, spo0A, codY, and SONO)were sub-cloned into the prototype vector pMTL5402FTTErmBtdRAM1, and theresultant recombinant plasmids introduced into the E. coli donor CA434,and thence used in conjugation experiments with either C. sporogenes orC. difficile as the recipient. In the case of the latter, notransconjugants were obtained. Transconjugants were obtained with bothplasmids in the case of C. sporogenes. Single transconjugants wereinoculated into 1.5 ml of an appropriate growth medium supplemented with250 μg/ml cycloserine and 7.5 μg/ml thiamphenicol (the latter of whichensures plasmid maintenance) and the culture was allowed to grow tostationary phase by anaerobic incubation at 37° C. overnight. 1500 ofthis culture was used to inoculate 1.5 ml of fresh broth of the sametype and containing the same supplements, which was then incubatedanaerobically at 37° C. As soon as growth was visible in the culture,typically after 1 hr, the culture was induced with 1 mM IPTG andincubated for 1 hr.

2 ml of the induced cells were harvested by centrifugation for 1 minuteat 7000 rpm, washed by re-suspension in PBS and harvested as before. Thepellet was re-suspended in an equal volume (2 ml) of an appropriategrowth medium without supplements, and incubated anaerobically at 37° C.for 1 hour. Serial dilutions of the culture were then plated onto anappropriate solid growth media supplemented with 1-10 μg/mlerythromycin, after 1 hr, 24 and 48 hr and incubated anaerobically at37° C.

No erythromycin colonies were obtained after two independent attempts.

EXAMPLE 8 Evidence that the Natural ErmB Promoter is Too Weak to DriveExpression of ErmB Sufficient for It to Act as a Selectable Marker

One explanation for the inability to detect retargeting of theretargeting nucleic acids as described in Example 7 is that the ermBpromoter is too weak to allow a single copy of the gene in a cell'schromosome to confer resistance to erythromycin. Analysis of the ermBpromoter sequence of Enterococcus faecalis plasmid pAMβ1 showed that thespacing between the promoter's −35 and −10 regions is 21 bp. The optimumfor Gram-positive promoters is 17±1 bp.

The ErmBtdRAM1 SE was cloned in two different orientations in pMT5402Frelative to fac. Only when the gene was under the control of fac was theplasmid carrying ErmBtdRAM1 SE capable of endowing the C. sporogeneshost with resistance to erythromycin. In the opposite orientation,transcription of the ermB coding region is reliant on its own promoterand expression was insufficient for resistance, despite ermB beingpresent on a multi-copy plasmid.

EXAMPLE 9 Development of a Clostridial ErmBtdRAM with a Strong Promoter

The promoter of the thl gene of C. acetobutylicum is recognised as astrong and constitutive promoter. Primers were designed to replace theErmBtdRAM1 promoter with the thl promoter. These delete unnecessarysequences between the transcriptional start site and ribosome bindingsite, and insert an NdeI site at the start codon to allow the promoterto be easily changed again if necessary. To guard against thepossibility that the thl promoter might be too strong, a mutant thlpromoter ‘thl2’ was also designed, by changing the spacing between the−35 and −10 to 16 nt, and making minor changes to the −35 and −10elements. Sequences of the thl and thl2 promoters spanning the −35 and−10 elements compared to a consensus Gram positive vegetative promoterare shown in FIG. 9. The sequence of the complete thl promoter is givenat positions 15-84 of the ErmBtdRAM2 sequence in FIG. 10. The sequenceof the complete thl2 promoter differs from the thl promoter only in theregion depicted in FIG. 9.

The thl and thl2 promoters were fused to the ErmBtdRAM1 and ErmBtdRAM1SE start codons using SOEing PCR and cloning steps, producing ErmBtdRAM2and ErmBtdRAM2 SE, each containing the thl promoter, and ErmBtdRAM3 andErmBtdRAM3 SE each containing the thl2 promoter. Sequence-correct cloneswere obtained of both RAM2 and RAM3, and were sub-cloned into pACD2 andpMTL20lacZTT for evaluation. The features and sequence of ErmBtdRAM2 isdepicted in FIG. 10.

The ability of the RAM2 and RAM3 portions to confer resistance toerythromycin on E. coli TOP10 cells was determined for plasmidscontaining these portions as indicated in Table 6 below.

TABLE 6 Erythromycin sensitivity of TOP10 clones bearing new constructsRAM TOPO SE TOPO pACD2::RAM pMTL20-lacZTTRAM pMTL5402F-TTRAM ErmBtdRAM2R R S S S ErmBtdRAM3 S R S S S

In all but the SE TOPO plasmid, the ermB gene is disrupted by the groupI intron, and therefore resistance was not expected. In the SE TOPOplasmid, the ermB gene is not disrupted, and so if the promoter of ermBis sufficiently strong, a resistance phenotype should be obtained.Unexpectedly, RAM2 TOPO clone conferred resistance to erythromycin in E.coli. It would appear that the very strong thl promoter and very highcopy number seems to overcome the presence of the group I intron in thiscontext, presumably by rare translation initiation at the, native ATG,This effect is only seen when the gene is present in TOPO. When insertedin a plasmid relevant for retargeting, such as pACD2 andpMTL20lacZTTRAM, E. coli cells are not resistant to erythromycin. TheRAM3 resistance profile was as expected. Therefore, either promoterappeared to be useful to drive expression of the selectable marker inthe Clostridial retargeting nucleic acid.

EXAMPLE 10 Evaluation of ErmBtdRAM2 and 3 in pACD2/pBRR3 System

The retargeting efficiency of the ErmBtdRAM2 and 3 were evaluated usingthe retargeting assay described in Example 5. Results are indicated inTable 7 below.

TABLE 7 Results of intron mobility assay Results Intron mobility RAMDonor plasmid efficiency* splicing efficiency† Shown previously: pACD2~10⁰    n/a pACD2KanRAM ~10⁻³ 18/20 pACD2ErmBtdRAM1 ~10⁻³ 18/20 Thisexperiment: pACD2ErmBtdRAM2 ~5 × 10⁻³  9/10 pACD2ErmBtdRAM3 ~7 × 10⁻² 9/10 *Intron mobility efficiency = Tet^(R) colonies/Amp^(R) Cm^(R)colonies †RAM splicing efficiency = Kan^(R) or Erm^(R) re-streakedTet^(R) colonies/all re-streaked Tet^(R) colonies

Splicing of RAM2 and RAM3 was efficient (90%), and equivalent to theoriginal RAM (RAM1).

EXAMPLE 11 Evaluation of ErmBtdRAM2 and 3 in pMTL20lacZTT System in E.coli

As previously, neither RAM could be used to detect retargeting of theGroup II intron into lacZ in the E. coli HMS174(DE3) chromosome usingeither Ery₅₀₀ or Ery₁₂₅. RAM2 but not RAM3 gave numerous Ery^(R)colonies, but they were shown by PCR not to contain a Group II intronretargeted to the lacZ gene. Presumably these colonies arose due to weakresistance conferred by the plasmid.

EXAMPLE 12

FIG. 11 illustrates the essential components of the vector pMTL007, alsoreferred to as pMTL5402FlacZTTErmBtdRAM2. This Group II intron ismodified to retarget the lacZ gene. It may be modified to retarget agene of a bacterial cell of the class Clostridia.

The essential elements of the plasmid are:

A clostridial promoter to bring about expression of the retargetingnucleic acid element, which in the illustrated example is the induciblefac promoter. Other promoters may be similarly employed which have beenmade inducible by provision of a lac operator, eg., fac2. To mediateinduction the plasmid also carries the E. coli lad gene under thecontrol of a clostridial promoter, in this instance the promoter of theptb gene (encoding phosphotransbutyrylase) of Clostridiumacetobutylicum. A constitutive promoter may be used instead of aninducible promoter. The plasmid also carries the ColE1 replicon ofplasmid pMTL20E, to allow maintenance of the plasmid in E. coli and thereplication region of the Clostridium butyricum plasmid pCB102, to allowmaintenance in Clostridium species. Maintenance of the plasmid is alsoprovided by the inclusion of the catP gene to enable selection of theplasmid in E. coli (through supplementation of the media withchloramphenicol) and Clostridia (through supplementation of the mediawith thiamphenicol). To provide the facility to conjugate the plasmidinto clostridial recipients in addition to transformation, the vectoralso carries the oriT region of plasmid RP4.

All of these elements are interchangeable with other equivalent factorsfrom other sources. Thus, ColE1 maybe exchanged with other repliconscapable of replication in E. coli, such as p15a, pVW01 or phage originssuch as M13. The catP gene may be substituted with other appropriateantibiotic resistance genes, such as tetM or aad. Similarly, anyreplicon capable of replicating in the targeted clostridial orGram-positive bacterial host may be employed, such as pIM13, pIP404,pAMB1, pCD6, pC194, pE194, pT181, pCB101, pBP1. Replicons which aredefective for replication may also be employed, including replicons thatmay be conditional for replication, eg., temperature sensitive orreliant on an exogenous factor for replication. Plasmids may also beemployed which lack any provision for replication in a Gram-positiveplasmid, ie., a suicide vector carrying only a ColE1 replicon

Other combinations of operator and repressor gene may be employed. Apromoter identified on the conjugative transposon Tn5397 which isregulated by tetracycline (Tet), represents another candidate (Roberts,PhD thesis, UCL). Alternatively, a xylose-inducible promoter, derivedfrom S. xylosus, has recently been shown to function in C.acetobutylicum (Girbal et al (2003) Appl Environ Microbiol. 69: 4985-8).Another candidate is a tet-regulated promoter developed in B. subtilis(Geissendorfer and Hillen (1990) Appl. Microbiol. Biotechnol. 33:657-63). It was constructed by adding a tet operator (tetO) sequencebetween the −35 and −10 of a strong xyl promoter (Geissendorfer andHillen, 1990, supra). In the presence of a tetR gene (encoding therepressor), the derivatised promoter was 100-fold inducible bysub-lethal concentrations of Tet. The basal levels of expressionobtained could be completely abolished by the addition of a second tetoperator, although this addition caused an overall reduction inexpression levels. Subsequently, this promoter has found wideapplication in S. aureus (Bateman et al (2001) Infect Immun. 69: 7851-7;Ji et al (2001) Science 293: 2266-2269), where only a single operatorproved necessary.

A tet-regulated promoter makes an ideal alternative to our developedfac/lacI system. Thus, we will be able to express tetR using the samepromoter used to express lacI (the C. acetobutylicum ptb promoter). InB. subtilis, the degree of induction was dose-dependent over the rangetested. However, as B. subtilis was sensitive to the antibiotic, highconcentrations of Tet could not be added. A similar constraint will notapply to clostridia such as C. difficile, which are resistant to thisantibiotic. To test the feasibility of the system, we will re-synthesisefac, replacing the region between the −35 and −10 with the tetO. Shouldhigh basal levels be observed in the absence of Tet, then a secondoperator can be added. Addition of further synthetic lacO sequences canalso be used to enhance repression of promoters by LacI (Muller et al(1996) J Mot Biol. 257: 21-9).

Construction of pMTL007

Oligonucleotide primers used in the construction are indicated in Table8 below.

TABLE 8 Oligonucleotide primers SEQ Primer Sequence (5′-3′) ID No.lacI-P1 GTGGTGCATATGAAACCAGTAACG 70 lacI-P2GAATTCCTAACTCACATTAATTGCGTTGCG 71 ptb-P1 GAATTCAGGGAATTAAAAGAATGTTTACCT72 G ptb-P2 ACTCATATGTTGCACCTCTACTTTAATAAT 73 TTTTAAC tdGpI-F1GCATTATGTTCAGATAAGGTCGTTAATCTT 29 ACCCC CatPFwdCAGCTGACCGGTCTAAAGAGGTCCCTAGCG 74 CC CatPSOER CGGTCATGCTGTAGGTACAAGGTAC75 CatPRev CAGCTGACCGGTCTCTGAAAATATAAAAAC 76 CACAGATTGATAC CatPSOEFGTACCTTGTACCTACAGCATGACCG 77 Thio-F1 CTACTAGTACGCGTTATATTGATAAAAATA 78ATAATAGTGGG Thio-R-RAM CCTTATCTGAACATAATGCCATATGAATCC 79CTCCTAATTTATACGTTTTCTC

A 1.627 kb LspI-HindIII fragment was isolated from the Clostridiumbutyricum plasmid pCB102 (Minton and Morris (1981) J Gen Microbiol 127:325-33) and blunt-ended with Klenow polymerase. The replicon cloningvector pMTL21E (Swinfield et al (1990) Gene 87:79-89) was cleaved withNheI, blunt-ended with Klenow polymerase and ligated with the isolatedpCB 102 replicon fragment. The resultant plasmid was designated pMTL540E(T Davis, PhD Thesis, The Open University, 1989), as shown in FIG. 12

The inducible promoter element was derived from the promoter of theferredoxin gene of Clostridium pasteurianum. Now termed fac, it wascreated by adding an E. coli lac operator immediately after the +1 ofthe ferredoxin gene promoter, and altering the sequence immediatelypreceding the ATG start codon of the ferredoxin structural gene to CAT,thereby creating a NdeI restriction site (CATATG) (Minton et al (1990)Vector systems for the genetic analysis of Clostridium acetobutylicumIn: Anaerobes in Human Medicine and Industry (eds P Boriello & JHardie), Wrightson Publishing, Petersfield, UK pp. 187-206). In thisparticular instance the lac operator inserted in the oppositeorientation relative to transcription compared to the lac promoter.However, this does not affect functionality, and Lad protein will stillbind and repress transcription from the promoter.

The fac promoter was then sub-cloned as an NdeI and EcoRI restrictionfragment, between the equivalent sites of plasmid pMTL1003 (Brehm et al(1991) Appl. Biotechnol. 36, 358-363), generating plasmid pMTL1006. Thissub-cloning step effectively removed the tip promoter of pMTL1003 andplaced the expression of iacZ' under the control of the modified fdpromoter. Plasmid pMTL1006 was then subjected to a BglI digest and thelarger of the two resultant fragments isolated. Plasmid pMTL500E(Oultram et al (1988) FEMS Microbiol Letts 56: 83-88) was similarlycleaved with BglI and the larger of the two fragments isolated andligated with the larger fragment isolated from pMTL1006. The plasmidobtained was designated pMTL500F (Fox et al, 1996, supra).

Although pMTL500F is replication proficient in clostridia, we have foundthat the transfer frequency of pAMβ1 based shuttle vectors intoclostridia is relatively inefficient. We therefore elected to change thereplicon to that of pCB102. Accordingly, both pMTL540E and pMTL500F werecleaved with BglI, and the larger fragment of pMTL540E ligated to thesmaller fragment of pMTL500F. The plasmid obtained was designatedpMTL540F (Fox et al, 1996, supra). For simplicity, the ligation ofpMTL500E and pMTL1006 fragments, and the ligation of pMTL540E andpMTL500F fragments is represented as a single ligation of pMTL540E andpMTL1006 in FIG. 12.

To enable conjugative transfer of the plasmids for those instances wheretransformation has yet to be demonstrated, we elected to endow theplasmid with the oriT (origin of transfer) of plasmid RP4. As such, theRP4 oriT region was excised from pEoriT (Purdy et al., 2002) using EcoRVand SmaI, and sub-cloned into the EcoRV restriction site of pMTL540F,generating pMTL5400F (see FIG. 12).

To bring about the production of LacI repressor protein, a promoter-lesscopy of the E. coli lad gene was amplified from pNM52 (Gilbert et al(1986) J. Gen. Microbiol. 132: 151-160) as an approx 1.0 kb NdeI-EcoRIfragment using the PCR primers lacI-P1 and lacI-P2 In parallel, thepromoter region of the Clostridium acetobutylicum ptb(phosphotransbutyrylase) gene was PCR amplified using the primers ptb-P1and ptb-P2. This localised the gene to a 578 by EcoRI-NdeI fragment. Thetwo fragments were isolated and ligated with EcoRI-cleaved pMTL20E,thereby placing the lad gene under the transcriptional control of theptb promoter, and localizing the modified gene to a portable EcoRIfragment. This fragment was excised from the plasmid generated,blunt-ended with Klenow polymerase, and ligated with EcoRV-cleavedpMTL5400F. The plasmid obtained was designated pMTL5401F, as shown inFIG. 12.

Plasmid pMTL5401F carries an erm gene as the selectable marker. It is,therefore, not compatible with the ErmRAM. The erm gene was thereforereplaced with the catP gene of pJIR418 (Sloan et al (1992) Plasmid 27:207-219). This was achieved by cleaving pMTL5401F with AhdI/TthIII1,blunt-ending the DNA with Klenow polymerase, and then ligating to a 1.1kb PvuII fragment carrying the pJIR418 catP gene to the larger of thetwo pMTL5401F fragments generated by cleavage with AhdI and TthIII1.This manipulation resulted in the complete deletion of ermB and removalof the majority of the bla gene. The plasmid obtained was designatedpMTL5402F, as shown in FIG. 13.

Prior to this substitution, a BsrG1 site within the catP fragment wasremoved by mutating a sequence to destroy the BsrG1 palindrome withoutchanging the catP coding sequence. This was undertaking using SewingOverlap Extension (SOE) PCR (Horton et al., 1990), using the primersCatPSOEF and CatPSOER. In addition, the flanking primers CatPFwd andCatPRev used were designed to encompass both a PvuII site and internalAgel sites. The former were incorporated for the subsequent insertion ofthe plasmid into pMTL5401F, whereas the latter were introduced tofacilitate the subsequent substitution of catP in the final plasmid,pMTL007, with alternative markers at a future date.

pMLT007 was constructed as follows:

The Targetron™ plasmid pACD4K-C was purchased from Sigma, andre-targeted to the E. coli lacZ gene using the control primers providedin the kit according to the provided protocol, except that the PCRproduct was first cloned and its sequence verified before sub-cloningthe HindIII/BsrGI fragment into pACD4K-C.

The lacZ-retargeting nucleic acid region was excised as a 5099 bp NaeIfragment and ligated into a 2412 bp fragment of pMTL20 which hadpreviously been generated by digestion with HindIII and SmaI, with T4polymerase blunting of the HindIII end, as shown in FIG. 13. A constructwas chosen in the orientation in which the HindIII and NheI sitesflanked the retargeting nucleic acid region.

The KanRAM was excised using MluI, and replaced with a 1259 bp MluIfragment containing ErmBtdRAM2, as shown in FIG. 13.

The entire lacZ-retargeting nucleic acid region including the ErmRAM wasthen excised as a ˜3.3 kbp HindIII/SacI fragment and a ˜1.8 kbpSacI/NheI fragment, which were ligated together into pMTL5402F digestedwith HindIII and NheI. The resulting plasmid was designatedpMTL5402FLacZTTErmBtdRAM1.

The thl promoter of C. acetobutylicum ATCC 824 was PCR-amplified frompSOS95 (Tummala et al (2003) J. Bacteriol. 185: 1923-1934) using primersThio-F1 and Thio-R-RAM. The PCR product was gel-purified and used, alongwith the td group I intron PCR product from the construction ofErmBtdRAM1, as template in a SOEing PCR using the outer primers Thio-F1and tdGpI-R1. The thl promoter and part of the td intron were excisedfrom this PCR product as a 143 bp SpeI/NspI fragment. The remainder ofthe td intron, and the ermB ORF from pCR2.1::ErmBtdRAM1 were excisedtogether as a NspI/NotI fragment. These fragments were ligated in athree-way ligation into pCR2.1::ErmBtdRAM1SE linearised with SpeI andNotI, yielding plasmid pCR2.1::ErmBtdRAM2.

The Mlu1/Mlu1 fragment of pCR2.1::ErmBtdRAM2 containing the RAM wasligated with the larger Mlu1/Mlu1 fragment of pMTL20lacZTT to formpMTL20lacZTTErmBtdRAM2 as shown in FIG. 14.

A BsrGI/BstBI fragment of pMTL20lacZTTErmBtdRAM2 containing the RAM wassubcloned into BsrGI/BstBI cleaved pMTL5402FlacZTTErmBtdRAM1 to generatepMTL007 as shown in FIG. 14.

pMTL007 was initially designated pMTL5402FlacZTTErmBtdRAM2, andsometimes referred to as pMTL5402FlacZTTErmRAM2 or pMTL5402FlacZTTRAM2or pMTL5402FlacZTTR2.

Once re-targeted, the plasmid was designated pMTL007 (orpMTL5402FTTErmBtdRAM2 or pMTL5402FTTErmBRAM2 or pMTL5402FTTRAM2 orpMTL5402FTTR2) suffixed by an identifier for the ‘Targeting Region’(TR). The TR is the entire region between the HindIII and BsrGI sites ofthe sequence generated by the re-targeting PCR. For example, once theplasmid was re-targeted to the C. difficile 630 gene spo0A, at position178 of the spo0A ORF, by cloning the appropriate TR fragment in as aHindIII/BsrGI fragment, the plasmid was designatedpMTL5402FTTErmBtdRAM2::Cd-spo0A-178aTR.

EXAMPLE 13 Evaluation of ErmBtdRAM2 and 3 in pMTL5402FlaZTT System in C.sporogenes Against codY

Having generated new RAMs that were capable of giving erythromycinresistance in Clostridia, Clostridial retargeting nucleic acidscomprising a modified Group II intron having targeting portions designedto target the intron to C. sporogenes against codY were constructed. Twoplasmids were constructed bearing either the RAM2 or the RAM3 and namedpMTL5402FCs-codY-417sTT::RAM2 and RAM3 respectively. The RAM2 version isidentical to that depicted in FIG. 10, except the retargeting portionsof the Group II intron and the IBS sequence are designed to allowretargeting of C. sporogenes against codY instead of E. coli lacZ.Either plasmid was conjugated into Clostridium sporogenes.Transconjugants were verified by PCR and shown to be completelysensitive to Ery_(1.25). A selected transconjugant of each RAM was theninduced with IPTG, and after removal of inducer by centrifugation andwashing, allowed 3 hours recovery before plating out on agar platescontaining a range of concentrations of erythromycin.

The number of colonies obtained is shown in Table 9 below.

TABLE 9 Results of retargeting assay Conditions Recov- Induc- ery tion(after Colonies per 100 μl 10⁰ plate (1 mM PBS Ery10 Ery5 Ery2.5 Ery1.25Expt RAM IPTG) wash) 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h A RAM2 1 h3 h 0 0 0 1 0 0 0 1 B RAM2 3 h 3 h 0.2 1 1 ~10 2 ~20 3 ~40 C RAM3 3 h 3h 0 0.2 0 0.2 0 0.6 0 0.6

These data demonstrate the importance of a sufficient induction period,and the superior efficiencies achieved using RAM2 compared to RAM3.

EXAMPLE 14 Further Mutant Generation

We elected to target two genes whose inactivation would lead to easilydetectable phenotypes. These were pyrF and spo0A. Inactivation of theformer should lead to uracil auxotrophy, while disruption of the lattershould lead to asporogeny.

pMTL007 was re-targeted to the C. sporogenes spo0A gene and hundreds ofEm^(R) colonies of C. sporogenes were readily obtained after IPTGinduction. DNA was extracted from four random colonies, and used as atemplate in PCR. In all cases, primers specific to the RAM generated aDNA fragment of a size consistent with loss of the td intron (FIG. 15).

Having demonstrated apparent functionality of the RAM withpMTL007::Csp-spo0A-249s, we proceeded to generate mutants in the twogenes (pyrF and spo0A) in all three clostridial species using theprotocols outlined in the methods section. PCR screening of Em^(R)clones (FIG. 15 b, c) revealed very high frequencies of insertion intothe intended chromosomal site (Table 10), demonstrating how easilyintegrants can be obtained using this method. After isolation, singlecolonies of integrants were screened for plasmid loss bythiamphenicol-sensitive phenotype, and colonies cured of the plasmidwere found to predominate in all these organisms without additionalpassaging. Insertion sites were verified by sequencing across theintron-exon junctions (Table 10) and Southern blotting with a probe forthe RAM confirmed the presence of a single copy of the insertion element(FIGS. 15 d, e and f).

IPTG induction of intron expression from pMTL007::Csp-spo0A-249s in C.sporogenes increased the insertion frequency by over 100-fold (Table10), in keeping with the reporter data from pMTL5401Fcat.

TABLE 10 Effect of regulated intron expression on insertion frequenciesin C. sporogenes. Relative insertion Plasmid IPTG^(a)frequInsertionency^(b) frequency^(c) pMTL007::Csp-spo0A-249s − ≦1.31 ±0.34 × 10⁻⁹ 1 pMTL007::Csp-spo0A-249s +    1.63 ± 0.72 × 10⁻⁷ 124pMTL007::Csp-spo0A- −    1.95 ± 0.54 × 10⁻⁶ 1489 249sΔlacI ^(a)Intronexpression was induced with 1 mM IPTG (+) or with water in place of IPTG(−). ^(b)After the recovery period, cells were spread onto TYGcycloserine plates with or without erythromycin supplementation.Insertion frequencies are expressed as Em^(R) c.f.u./ml/total c.f.u./ml.^(c)Relative insertion frequencies are normalised to the experiment withpMTL007::Csp-spo0A-249s and water in place of IPTG.

To establish whether regulated expression of the intron conferred anyadvantage over constitutive expression, we de-repressed the fac promoterby introducing a frameshift mutation into the lacI gene ofpMTL007::Csp-spo0A-249s. A further insertion frequency increase of over10-fold was observed (Table 10), indicating that regulated expression ofthe intron confers no advantage over constitutive expression. Weperformed an equivalent experiment in C. acetobutylicum withpMTL007::Cac-spo0A-242a and observed no change in integrationfrequencies with the addition of IPTG (data not shown). Consistent withthe pMTL5401Fcat reporter data, basal intron expression from the facpromoter in this organism is apparently sufficient to achieveeasily-detectable integration frequencies. Like pMTL5401F, pMTL007 istoo unstable in C. difficile to support the growth of itstransconjugants in antibiotic-supplemented liquid culture (Purdy et al(2002) Mol. Microbiol. 46: 439-452). Therefore no comparableIPTG-induction experiments to those undertaken in C. sporogenes could beperformed. However, in both C. difficile and C. acetobutylicum, Em^(R)integrants could be easily obtained by simply re-streakingtransconjugant colonies onto growth media containing erythromycin withno addition of IPTG.

As anticipated, all the spo0A mutants were unable to form endospores(FIG. 16). All of the pyrF mutants were shown to be unable to grow onminimal media unless supplemented with 50 μg/L uracil. We attempted toselect revertants to uracil prototrophy by growing all three clostridialmutants in rich liquid media lacking erythromycin selection and thenplating them onto minimal agar medium with or without uracil. Revertantswere never detected on media lacking uracil in at least threeexperiments. By comparison to the cell counts on media supplemented withuracil, reversion frequencies per cell were estimated to be less than9.36×10⁻⁹ in C. difficile, less than 9.60×10⁻⁷ in C. acetobutylicum andless than 5.50×10⁻⁹ in C. sporogenes. These findings are consistent withdata in the literature (Frazier et al (2003) Appl. Environ. Microbiol.69: 1121-1128) showing that intron integrants are extremely stable—ahighly desirable mutant characteristic.

EXAMPLE 15 Evaluation of ErmBtdRAM2 System Against Other Targets

A standard protocol has been developed for retargeting in Clostridia, asfollows.

1. Intron Re-Targeting Sequences to the Gene of Interest are GeneratedEssentially According to the Method Provided by Sigma with theTargetron™ Kit:

The computer algorithm provided at the Sigma website[http://www.sigma-genosys.com/targetron/] is used to identify possibleintron targets within the sequence of the gene of interest, and todesign PCR primers. These primers are then used according to the SigmaTargetron™ protocol, and using PCR reagents provided in the SigmaTargetron™ kit, to generate a 353 bp PCR product which corresponds topart of the intron and includes modified IBS, EBS1d and EBS2 sequencessuch that the intron can be re-targeted to the gene of interest. ThisPCR product is cloned into an appropriate cloning vector such as pCR2.1and its sequence verified. Alternatively, it may be subcloned directlyinto pMTL007.

2. The Prototype Clostridial Retargeting Plasmid pMTL5402FlacZTTR2 isRe-Targeted Essentially According to the Method Provided by Sigma withthe Targetron™ Kit:

If the PCR product of step 1 was cloned into a cloning vector, thedesired re-targeting sequence is excised from its plasmid by digestionwith the restriction enzymes HindIII and BsrGI, and cloned intopMTL5402FlacZTTR2 digested with the same enzymes. In either case, theresultant constructs are verified by restriction analysis and/orsequencing.

3. The Successfully Re-Targeted Clostridial Retargeting Plasmid isTransferred into the Target Organism:

Recombinant plasmids may be introduced into the clostridial hosts bystandard DNA transfer methods based either on electrotransfoimation orconjugation. Methods for either are given in Davis I, Carter G, Young Mand Minton N P (2005) “Gene Cloning in Clostridia”, In: Handbook onClostridia (Dune P, ed) pp. 37-52, CRC Press, Boca Raton, USA. In ourexperiments, plasmids were introduced into Clostridium difficile andClostridium sporogenes by conjugation from E. coli donors. In contrast,plasmids were introduced into Clostridium acetobutylicum bytransformation.

4. Retargeting Nucleic Acid Expression and Subsequent Integration isAchieved by Induction of the Transformant with IPTG:

An individual transformant colony is used to inoculate 1.5 ml of anappropriate growth medium supplemented with 250 μg/ml cycloserine and7.5 μg/ml thiamphenicol (the latter of which ensures plasmidmaintenance) and the culture is allowed to grow to stationary phase byanaerobic incubation at 37° C. overnight. 150 μl of this culture is usedto inoculate 1.5 ml of fresh broth of the same type and containing thesame supplements, which is then incubated anaerobically at 37° C. Assoon as growth is visible in the culture, typically after 1 hr, theculture is induced with 1 mM IPTG and incubated for 3 hrs.

5. Retargeting Nucleic Acid Integrants are Detected and Isolated Using aRecovery Step Followed by Plating of Cells onto Selective Solid Mediaand Incubation:

2 ml of the induced cells are harvested by centrifugation for 1 minuteat 7000 rpm, washed by re-suspension in PBS and harvested as before. Thepellet is re-suspended in an equal volume (2 ml) of an appropriategrowth medium without supplements, and incubated anaerobically at 37° C.for 3 hrs. Serial dilutions of the culture are then plated onto anappropriate solid growth media supplemented with 1-10 μg/mlerythromycin, and incubated anaerobically at 37° C. Erythromycinresistant colonies corresponding to retargeting nucleic acid integrantclones can be picked from these plates after 18-48 hrs, depending uponthe organism and erythromycin concentration used.

Optionally, serial dilutions of the culture can additionally be platedonto unsupplemented solid growth media or solid growth mediasupplemented with 15 μg/ml thiamphenicol in place of erythromycin inorder to determine the frequency of the integration event.

The standard protocol was used to make Clostridial mutants as indicatedin Table 11.

TABLE 11 Clostridial mutants Organism Target Re-Targeted^(a) PercentageIn Target Gene^(b) C. sporogenes codY YES Not yet determined C.sporogenes spo0A YES 100% (3 of 3) C. sporogenes pyrF YES 100% (2 of 2)C. acetobutlyicum pyrF YES Not yet determined C. difficile spo0A YES100% (3 of 3) Diagnostic PCR primers give a product of the expected sizeif the retargeting nucleic acid has inserted in the targeted gene.^(a)Presence of desired mutant demonstrated in a pool of several clones^(b)Several individual clones screened for desired mutation

Sometimes, retargeting is inefficient. Therefore, it is recommended totry more than one targeting portion to disrupt any given gene.Furthermore, colonies may be pooled before PCR screening of combinedbatches. If, say portions of 10 or 100 colonies were combined and a PCRproduct of the size expected for a retargeted mutant was generated,colonies could then be individually screened.

EXAMPLE 16 Further Mutant Generation

To further establish the utility of the method, we selected severalother genes from each of the three species, and repeated the mutagenesisprocedure. The genes targeted are listed in Table 12 and theoligonucleotide primers used to generate PCR products according to thestandard protocol for modification of the Group II intron of pMTL007 areshown in Table 4 or Table 13. In every case the desired integrant wasobtained. Each insertion was confirmed by PCR screening and theinsertion point verified by nucleotide sequencing.

TABLE 12 Intron insertion frequencies with erythromycin selection Em^(R)Desired Frequency of Target (Organism, clones mutants desired mutantInsertion site SEQ ORF and insertion point^(a)) screened^(b)obtained^(b) among Em^(R )clones^(b) verified by sequencing^(b) ID NoC. sporogenes spo0A 249s  4 4 100% TATGCCAAGG-intron-GTAATTGTTT 80C. difficile spo0A 178a  3 3 100% ATTACATCTA-intron-GTATTAATAA 81C. acetobutylicum spo0A 242a  8 4  50% TATTCTTGGA-intron-AGGTTTTCTG 82C. sporogenes pyrF595s  2 2 100% ATAGGAGCAG-intron-TAGTTGGATG 83C. difficile pyrF97a 96^(c) 7-19^(c) 7-20%^(c)ACCTTAAATA-intron-TGTCTACACT 84 C. acetobutylicum pyrF 345s  8 2  25%CTTTGAAGGT-intron-GATTTTGAAG 85 C. acetobutylicum CAC0081 141a  6 6 100%TTTTAATGAC-intron-ATAGTTTATA 86 C. acetobutylicum CAC0080 121s  6 6 100%CTGAAATTAT-intron-TTCGTTAATA 87 C. acetobutylicum CAC0078 385a  3 3 100%GTATCTCCAG-intron-GCGCATATCT 88 C. acetobutylicum CAC2208 201s  4 3  75%TGTGGAGTAT-intron-TCGGTACACA 89 C. difficile CD0153 784a  5 5 100%CCAATAAGCC-intron-CATCTCCAGA 90 C. difficile CD0552 75a 10 9  90%CTCTACAATA-intron-TCTATCTTTA 91 C. difficile CD3563 226s  8 8 100%GAGGGACAGG-intron-TTGCTGTAGC 92 C. botulinum CBO0780 671a  4 4 100%TTTATTATTT-intron-TCTTTTTTAA^(d) 93 C. botulinum CBO1120 670s  4 4 100%GAATTTTATG-intron-CTAATATATC^(d) 94 C. botulinum CBO2762 1014s  4 4 100%TTTAACATAT-intron-AGATTAGTTA^(d) 95 C. botulinum spo0A 249s  4 4 100%TATGCCAAGG-intron-GTAATTGTTT^(d) 96 ^(a)Introns were inserted after theindicated number of bases from the start of the ORF, in either the sense(s) or antisense (a) orientation. ^(b)Genomic DNA was extracted fromEm^(R) clones picked at random and used as template in PCR using primerswhich amplify across an intron-exon junction. One clone of each desiredmutant was selected and the intron insertion site verified bysequencing. ^(c)Ninety-six Em^(R) C. difficile pyrF mutant candidateclones were screened in pools. Exhaustive screening was not required toisolate the mutant, so a range of possible frequencies is given.^(d)Predicted site of insertion, not verified by nucleotide sequencing.

TABLE 13 Oligonucleotide primers Oligonucleotide Sequence (5′-3′)SEQ ID No Cdi-CD0552-75a-IBSAAAAAAGCTTATAATTATCCTTATTCTCCACAATAGTGCGCCCAGATAGGGTG  97Cdi-CD0552-75a-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCACAATATCTAACTTACCTTTCTTTGT  98Cdi-CD0552-75a-EBS2 TGAACGCAAGTTTCTAATTTCGGTTGAGAATCGATAGAGGAAAGTGTCT 99 Cdi-CD3563-226s-IBSAAAAAAGCTTATAATTATCCTTAATGAGCGACAGGGTGCGCCCAGATAGGGTG 100Cdi-CD3563-226s-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCGACAGGTTTAACTTACCTTTCTTTGT 101Cdi-CD3563-226s-EBS2 TGAACGCAAGTTTCTAATTTCGGTTCTCATCCGATAGAGGAAAGTGTCT102 Cac-CAC0081-141a-IBSAAAAAAGCTTATAATTATCCTTAATTTTCAATGACGTGCGCCCAGATAGGGTG 103Cac-CAC0081-141a-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCAATGACATTAACTTACCTTTCTTTGT 104Cac-CAC0081-141a-EBS2 TGAACGCAAGTTTCTAATTTCGGTTAAAATCCGATAGAGGAAAGTGTCT105 Cac-CAC0078-385a-IBSAAAAAAGCTTATAATTATCCTTACTGTACCTCCAGGTGCGCCCAGATAGGGTG 106Cac-CAC0078-385a-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCCTCCAGGCTAACTTACCTTTCTTTGT 107Cac-CAC0078-385a-EBS2 TGAACGCAAGTTTCTAATTTCGATTTACAGTCGATAGAGGAAAGTGTCT108 Cac-CAC0080-121s-IBSAAAAAAGCTTATAATTATCCTTAAACTGCAATTATGTGCGCCCAGATAGGGTG 109Cac-CAC0080-121s-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCAATTATTTTAACTTACCTTTCTTTGT 110Cac-CAC0080-121s-EBS2 TGAACGCAAGTTTCTAATTTCGGTTCAGTTCCGATAGAGGAAAGTGTCT111 Cac-CAC2208-201s-TBSAAAAAAGCTTATAATTATCCTTACATGTCGAGTATGTGCGCCCAGATAGGGTG 112Cac-CAC2208-201s-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCGAGTATTCTAACTTACCTTTCTTTGT 113Cac-CAC2208-201s-EBS2 TGAACGCAAGTTTCTAATTTCGGTTACATGTCGATAGAGGAAAGTGTCT114 Cdi-CD0153-784a-IBSAAAAAAGCTTATAATTATCCTTATACCACTAAGCCGTGCGCCCAGATAGGGTG 115Cdi-CD0153-784a-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCTAAGCCCATAACTTACCTTTCTTTGT 116Cdi-CD0153-784a-EBS2 TGAACGCAAGTTTCTAATTTCGGTTTGGTATCGATAGAGGAAAGTGTCT117 Cbo-CBO0780-671a-IBS1AAAAAAGCTTATAATTATCCTTAGCTTTCTTATTTGTGCGCCCAGATAGGGTG 118Cbo-CBO0780-671a-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCTTATTTTCTAACTTACCTTTCTTTGT 119Cbo-CBO0780-671a-EBS2 TGAACGCAAGTTTCTAATTTCGATTAAAGCTCGATAGAGGAAAGTGTCT120 Cbo-CBO1120-670s-IBS1AAAAAAGCTTATAATTATCCTTACTGAACTTTATGGTGCGCCCAGATAGGGTG 121Cbo-CBO1120-670s-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCTTTATGCTTAACTTACCTTTCTTTGT 122Cbo-CBO1120-670s-EBS2 TGAACGCAAGTTTCTAATTTCGGTTTTCAGTCGATAGAGGAAAGTGTCT123 Cbo-CBO2762-1014s-IBS1AAAAAAGCTTATAATTATCCTTAGATTTCACATATGTGCGCCCAGATAGGGTG 124Cbo-CBO2762-1014s-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCACATATAGTAACTTACCTTTCTTTGT 125Cbo-CBO2762-1014s-EBS2 TGAACGCAAGTTTCTAATTTCGATTAAATCTCGATAGAGGAAAGTGTCT126 Cbo-spo0A-249s-IBS1AAAAAAGCTTATAATTATCCTTACCTATCCCAAGGGTGCGCCCAGATAGGGTG 127Cbo-spo0A-249s-EBS1dCAGATTGTACAAATGTGGTGATAACAGATAAGTCCCAAGGGTTAACTTACCTTTCTTTGT 128Cbo-spo0A-249s-EBS2 TGAACGCAAGTTTCTAATTTCGGTTATAGGTCGATAGAGGAAAGTGTCT129

EXAMPLE 17 Construction of a catP-Based RAM

A RAM containing an alternative modified selectable marker gene, namelycatP, which confers resistance to chloramphenicol or thiamphenicol, wasconstructed.

The catP ORF was PCR-amplified from pMTL5402F plasmid DNA template usingprimers linker-catP-F5′-ATACTCAGGCCTCAATTAAC-CCAAGAGA-TGCTGGTGCTTCTGGTGCTGGTATGGTATTTGAAAAAATTGATAAAAATAGTTGGAACAG-3′(SEQ ID No. 130) and catP-MluI-R15′-ATACGC-GTTTAACTATTTATCAATTCCTGCAATTCGTTTACAAAACGGC-3 (SEQ ID No.131), which added a small part of the td intron and linker to the 5′ ofthe catP ORF using a primer extension.

The PCR product was digested with StuI and MluI. A portion of EnatdRAM2containing the thl promoter, linker and most of the td intron wasexcised as a SpeI/StuI fragment from pCR2.1::ErmBtdRAM2. These tworestriction fragments were ligated together into pCR2.1::ErmBtdRAM2linearised with SpeI and MluI, yielding the plasmid pCR2.1::RAM-C1,which contains the new RAM element RAM-C1.

The sequence immediately preceding the catP ORF in RAM-C1 is identicalto the sequence immediately preceding the ermB ORF in ErmBtdRAM2,containing the thl promoter, linker and td group I intron. The entireRAM-C1 element is flanked by MluI sites to facilitate its sub-cloninginto the MluI site of the L1.LtrB intron for use as a RAM.

The RAM-C1 or a derivative thereof may be used as the RAM element in aplasmid analogous to pMTL007 to select for retargeting events inClostridia on the basis of acquisition of thiamphenicol orchloramphenicol resistance. It will be appreciated that the selectablemarker that is required to maintain the plasmid in the host must conferresistance to a different agent from the resistance conferred by theRAM. Therefore, pMTL007 will be modified by replacement of its catPselectable marker with a different selectable marker, such as ermB,which is effective in Clostridia. A plasmid modified in this way may beused for retargeting Clostridia.

As described herein, the promoter operatively linked to the regionencoding the selectable marker must be capable of causing expression ofthe selectable marker encoded by a single copy of the selectable markergene in an amount sufficient for the selectable marker to alter thephenotype of the Clostridial cell such that it can be distinguished fromthe Clostridial cell lacking the selectable marker gene. If the thlpromoter in the RAM-C1 element fails to fulfil this criterion, it may bereplaced or modified using methods disclosed herein. Similarly, if thepositioning of the td group I intron is inappropriate either to preventexpression of the selectable marker when it is present in the RAM, or topermit expression of the selectable marker when it has spliced out ofthe RAM, its position may be modified. The function of the elements ofthe RAM may be tested using the two-plasmid system developed Karberg etal (2001) (see Example 5). Ultimately, RAM-C1, or a derivative thereof,will be used to generate retargeting mutants in Clostridia.

1. A DNA molecule comprising: a modified Group II intron which does notexpress the intron-encoded reverse transcriptase but which contains amodified selectable marker-gene in the reverse orientation relative tothe modified Group II intron, wherein the modified selectable markergene comprises a region encoding a selectable marker and a promoteroperably linked to said region, wherein the promoter causes expressionof the selectable marker encoded by a single copy of the modifiedselectable marker gene in an amount sufficient for the selectable markerto alter the phenotype of a bacterial cell of the class Clostridia suchthat it can be distinguished from the bacterial cell of the classClostridia lacking the modified selectable marker gene; and a promoterfor transcription of the modified Group II intron, said promoter beingoperably linked to said modified Group II intron; and wherein themodified selectable marker gene contains a Group I intron positioned inthe forward orientation relative to the modified Group II intron so asto disrupt expression of the selectable marker; and wherein the DNAmolecule allows for removal of the Group I intron from the RNAtranscript of the modified Group II intron to leave a region encodingthe selectable marker and allows for the insertion of said RNAtranscript (or a DNA copy thereof) at a site in a DNA molecule in abacterial cell of the class Clostridia. 2-39. (canceled)
 40. The DNAmolecule of claim 1, further comprising exons flanking the modifiedGroup II intron, wherein the exons allow splicing of an RNA transcriptof the modified Group II intron.
 41. The DNA molecule of claim 40,wherein the modified Group II intron further comprises targetingportions.
 42. The DNA molecule of claim 41, wherein the targetingportions guide insertion of the RNA transcript of the modified Group IIintron into a site within a DNA molecule in the bacterial cell of theclass Clostridia.
 43. The DNA molecule of claim 42, wherein the site isa selected site.
 44. The DNA molecule of claim 43, wherein the DNAmolecule is a plasmid.
 45. The DNA molecule of claim 44, wherein theplasmid is an Escherichia coli-Clostridia shuttle vector.
 46. The DNAmolecule of claim 45, further comprising a region permitting conjugativetransfer from Escherichia coli to a bacterial cell of the classClostridia.
 47. The DNA molecule of claim 1, wherein the promoteroperably linked to the region encoding the selectable marker is thepromoter of the thl gene of C. acetobutylicum, the ptb gene of C.acetobutylicum or the adc gene of C. acetobutylicum or the promoter ofthe fdx gene of C. perfringens or the cpe gene of C. perfringens. 48.The DNA molecule of claim 1, wherein the selectable marker confers agrowth advantage on a bacterial cell of the class Clostridia in whichthe selectable marker gene is expressed, compared to a bacterial cell ofthe class Clostridia lacking the selectable marker.
 49. The DNA moleculeof claim 48, wherein the selectable marker confers erythromycinresistance or chloramphenicol resistance to the bacterial cell of theclass Clostridia.
 50. The DNA molecule of claim 1, wherein the Group Iintron is located within or upstream of the region encoding theselectable marker.
 51. The DNA molecule of claim 1, wherein the promoteroperably linked to the modified Group II intron is an induciblepromoter.
 52. The DNA molecule of claim 51, wherein the induciblepromoter is inducible by isopropyl β-D-1-thiogalactopyranoside (“IPTG”)or xylose.
 53. The DNA molecule of claim 51, further comprising an openreading frame encoding a Group II intron-encoded reverse transcriptaseoperably linked to a promoter but not contained in the modified Group IIintron.
 54. The DNA molecule of claim 1, wherein the bacterial cell ofthe class Clostridia is of the genus Clostridium.
 55. The DNA moleculeof claim 54, wherein the bacterial cell of the genus Clostridium is C.thermocellum, C. acetobutylicum, C. difficile, C. botulinum, C.perfringens, C. beijerinckii, C. tetani, C. cellulyticum, or C.septicum.
 56. The DNA molecule of claim 43, wherein the selected site inthe DNA molecule in the bacterial cell of the class Clostridia islocated within a gene or within a portion of DNA which affects theexpression of a gene.
 57. The DNA molecule of claim 56, wherein the siteis located within the chromosomal DNA of the bacterial cell of the classClostridia.
 58. A kit comprising (i) the DNA molecule of claim 1 and(ii) a DNA molecule encoding a Group II intron-encoded reversetranscriptase.
 59. A method of splicing a nucleic acid molecule into asite in a DNA molecule in a bacterial cell of the class Clostridia, themethod comprising the steps of: (i) providing a bacterial cell of theclass Clostridia with the DNA molecule of claim 1 and a DNA moleculeencoding a Group II intron-encoded reverse transcriptase; and (ii)culturing the bacterial cell of the class Clostridia under conditionswhich allow for removal of the Group I intron from the RNA transcript ofthe DNA molecule of claim 1 and insertion of the RNA transcript intosaid site.
 60. The method of claim 59, further comprising culturing thebacterial cell of the class Clostridia under conditions in which theselectable marker is expressed.
 61. The method of claim 60, furthercomprising selecting the bacterial cell of the class Clostridia based onan altered phenotype conferred by the selectable marker.
 62. The methodof claim 61, further comprising the step of isolating a single clone ofcells derived from the bacterial cell of the class Clostridia.
 63. Themethod of claim 59, wherein the bacterial cell of the class Clostridiais of the genus Clostridium.
 64. The method of claim 63, wherein thebacterial cell of the genus Clostridium is C. thermocellum, C.acetobutylicum, C. difficile, C. botulinum, C. perfringens, C.beijerinckii, C. tetani, C. cellulyticum, or C. septicum.
 65. The methodof claim 59, wherein the site in the DNA molecule in the bacterial cellof the class Clostridia is located within a gene or within a portion ofDNA which affects the expression of a gene.
 66. The method of claim 65,wherein the site is located within the chromosomal DNA of the bacterialcell of the class Clostridia.
 67. The method of claim 59, wherein theproviding the bacterial cell of the class Clostridia with the DNAmolecule comprises transducing or transferring the DNA molecule into thebacterial cell of the class Clostridia, or transconjugating the DNAmolecule from a donor bacterial cell into the bacterial cell of theclass Clostridia.
 68. A method of targeting a nucleic acid molecule to aselected site in a DNA molecule in a bacterial cell of the classClostridia, the method comprising: (i) proiding a bacterial cell of theclass Clostridia with the DNA molecule of claim 41 and a DNA moleculeencoding a Group II intron-encoded reverse transcriptase; and (ii)culturing the bacterial cell under conditions which allow for removal ofthe Group I intron from the RNA transcript of the DNA molecule of claim41 and insertion of said RNA transcript containing the selectable markergene (or a DNA copy thereof) into said selected site.
 69. A methodaccording to claim 68, wherein the selected site of the DNA molecule isa gene or a portion of DNA which affects the expression of a gene.
 70. Amutant bacterial cell of the class Clostridia obtained by the method ofclaim
 59. 71. A mutant bacterial cell of the class Clostridia obtainedby the method of claim
 69. 72. A DNA molecule comprising a modifiederythromycin-resistance gene containing a Group I intron which disruptsexpression of the erythromycin resistance gene, wherein when the Group Iintron is transcribed it is able to excise itself from the RNAtranscript.
 73. A DNA molecule comprising a modifiedchloramphenicol-resistance gene containing a Group I intron whichdisrupts expression of the chloramphenicol resistance gene, wherein whenthe Group I intron is transcribed it is able to excise itself from theRNA transcript.
 74. A DNA molecule comprising a modifiedtetracycline-resistance gene containing a Group I intron which disruptsexpression of the tetracycline resistance gene, wherein when the Group Iintron is transcribed it is able to excise itself from the RNAtranscript.
 75. A DNA molecule comprising a modifiedspectinomycin-resistance gene containing a Group I intron which disruptsexpression of the spectinomycin resistance gene, wherein when the GroupI intron is transcribed it is able to excise itself from the RNAtranscript.
 76. The DNA molecule of claim 72, wherein the DNA moleculeis a plasmid.
 77. A host cell comprising a DNA molecule according toclaim
 76. 78. The DNA molecule of claim 73, wherein the DNA molecule isa plasmid.
 79. A host cell comprising a DNA molecule according to claim78.
 80. The DNA molecule of claim 74, wherein the DNA molecule is aplasmid.
 81. A host cell comprising a DNA molecule according to claim80.
 82. The DNA molecule of claim 75, wherein the DNA molecule is aplasmid.
 83. A host cell comprising a DNA molecule according to claim82.